Application of porous starch-self-assembled nano-carrier microcapsule

Abstract

The invention discloses an application of a porous starch-self-assembled nano-carrier microcapsule, which comprises the following steps: firstly, loading active substances by using protein and polysaccharide colloidal particles, and then filling the protein / polysaccharide particles loaded with polyphenol into porous starch as a whole, so as to obtain the porous starch-self-assembled nano-carrier microcapsule. A porous starch-self-assembled nano-carrier microcapsule drug delivery system for treating ulcerative colitis is prepared by using a self-assembled nano-carrier as a carrier, the dissolution of polyphenol in simulated gastric juice and intestinal juice is effectively inhibited, the digestion stability of polyphenol is improved, and finally, most polyphenol is delivered to a large intestine part, so that high-concentration continuous delivery of targeted colorectum is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of porous starch-self-assembled nanocarrier microcapsules. Background Art

[0002] Under environmental, genetic, immune, or bacterial stressors, the intestine is highly susceptible to inflammatory damage, leading to an increase in reactive oxygen species (ROS) in the intestine. Excessive ROS can cause local mucosal damage and amplify the inflammatory response. Polyphenols, including quercetin, curcumin, and resveratrol, are secondary metabolites with a wide range of physiological activities. Using these bioactive ingredients with antioxidant activity for antioxidant therapy is considered a potential strategy for alleviating intestinal inflammatory diseases. Polyphenols can modulate the intestinal microbiota, promote the growth of probiotics, and inhibit the proliferation of pathogenic bacteria, and have been shown to have multiple beneficial effects. However, polyphenols suffer from poor water solubility, sensitivity to light and heat, oxidative decomposition, and low bioavailability, which reduce their physiological efficacy and limit their application in food and medical fields. Therefore, improving the solubility, stability, and bioavailability of polyphenols and efficiently delivering them to specific locations to exert their functional properties are currently urgent issues to be addressed. Summary of the Invention

[0003] The present application provides an application of porous starch-self-assembled nanocarrier microcapsules in the preparation of drugs for treating ulcerative colitis. The porous starch-self-assembled polyphenol nanocarrier drug delivery system can effectively inhibit the dissolution of polyphenols in simulated gastric juice and small intestinal juice, provide digestive stability of polyphenols, and can be efficiently delivered to the large intestine to achieve high-concentration sustained delivery targeted to the colorectum, alleviate weight loss in individuals with colitis, alleviate colon shortening and colon damage caused by colitis in individuals, and reduce the level of intestinal inflammation in individuals with colitis.

[0004] Application of porous starch-self-assembled nanocarrier microcapsules in the preparation of drugs for preventing or treating ulcerative colitis. The preparation of the porous starch-self-assembled polyphenol nanocarrier drug delivery system comprises:

[0005] (1) adjusting the pH of a mixed aqueous solution containing sodium carboxymethyl cellulose (CMC) and ovalbumin (OVA) to acidic, and then heating the mixture in a water bath to react, thereby obtaining an OVA / CMC nanoparticle solution;

[0006] (2) adding a short-chain alcohol solution of polyphenols to the obtained OVA / CMC nanoparticle solution to obtain a polyphenol mixed solution, and evaporating the short-chain alcohol in the polyphenol mixed solution to obtain an OVA / CMC-Qu nanoparticle solution;

[0007] (3) The porous starch is uniformly dispersed in the aqueous solution of OVA / CMC-Qu nanoparticles for dynamic adsorption. After the adsorption is completed, the resulting reaction solution is filtered, and the filter residue is collected and dried.

[0008] This application first uses protein and polysaccharide colloidal particles to load active substances, and then loads the protein / polysaccharide nanoparticles loaded with polyphenols into porous starch as a whole to prepare a porous starch-self-assembled nanocarrier microcapsule drug delivery system for the treatment of ulcerative colitis, which effectively inhibits the dissolution of polyphenols in simulated gastric juice and small intestinal juice, provides digestive stability of polyphenols, and ultimately delivers most of the polyphenols to the large intestine, achieving high-concentration sustained delivery targeting the colorectum. The drug delivery carrier of this application can alleviate weight loss in individuals with colitis, alleviate colon shortening and colon damage caused by colitis in individuals, and reduce the level of intestinal inflammation in individuals with colitis. It effectively guarantees the application of polyphenols as oral nutritional supplements for intestinal inflammatory diseases and provides new ideas and theoretical guidance for the design, preparation and research of oral active substances or drug carriers.

[0009] In step (1):

[0010] Optionally, in the mixed aqueous solution, the mass percentage concentration of sodium carboxymethyl cellulose CMC is 1-3%, and the mass percentage concentration of ovalbumin OVA is 0.5-1.5%; further preferably, in step (1), in the mixed aqueous solution, the mass percentage concentration of sodium carboxymethyl cellulose CMC is 2%, and the mass percentage concentration of ovalbumin OVA is 1%.

[0011] Optionally, the pH is adjusted to 2-3; more preferably, the pH is adjusted to 2.

[0012] Optionally, the water bath temperature is 70-80°C; more preferably, the bath temperature is 80°C.

[0013] Optionally, the reaction time in the water bath is 20 to 40 minutes; more preferably, the reaction time in the water bath is 30 minutes.

[0014] In step (2):

[0015] Optionally, the polyphenol is at least one of quercetin, curcumin, and resveratrol; further preferably, the polyphenol is quercetin.

[0016] Optionally, the short-chain alcohol is methanol, ethanol or propanol; further preferably, the short-chain alcohol is ethanol.

[0017] Optionally, the concentration of the polyphenols in the short-chain alcohol solution of polyphenols is 5-15 mg / mL, and the volume ratio of the short-chain alcohol solution of polyphenols to the OVA / CMC nanoparticle solution is 1-5:50. Further preferably, the concentration of the polyphenols in the short-chain alcohol solution of polyphenols is 10 mg / mL, and the volume ratio of the short-chain alcohol solution of polyphenols to the OVA / CMC nanoparticle solution is 3:50.

[0018] In step (3):

[0019] Optionally, the concentration of the OVA / CMC-Qu nanoparticle aqueous solution is 0.01-15 mg / mL; the mass volume ratio of the porous starch to the OVA / CMC-Qu nanoparticle aqueous solution is 3-10 g:50 mL.

[0020] Furthermore, the concentration of the OVA / CMC-Qu nanoparticle aqueous solution is 0.1-5 mg / mL; more preferably, the concentration of the OVA / CMC-Qu nanoparticle aqueous solution is 0.5 mg / mL; the mass volume ratio of the porous starch to the OVA / CMC-Qu nanoparticle aqueous solution is 5 g:50 mL.

[0021] Optionally, the dynamic adsorption is performed under shaking or stirring conditions, and the adsorption time is 10 to 20 minutes. More preferably, the adsorption time is 15 minutes.

[0022] Optionally, the drug is used for at least one of the following:

[0023] Improve colon atrophy or colon damage caused by colitis;

[0024] Inhibit the production of pro-inflammatory factors in colon tissue;

[0025] Relieves weight loss in individuals with colitis.

[0026] Optionally, the pro-inflammatory factors include TNF-α and IL-6.

[0027] Optionally, the drug is an oral preparation, and the dosage form of the oral preparation is selected from tablets, liquids, powder granules or soft gels.

[0028] Optionally, the drug further contains pharmaceutically acceptable excipients.

[0029] Compared with the prior art, this application has at least one of the following beneficial effects:

[0030] (1) The porous starch-self-assembled polyphenol nanocarrier drug delivery system of the present application can effectively inhibit the dissolution of polyphenols in simulated gastric juice and small intestinal juice, provide digestion stability of polyphenols, and ultimately deliver most of the polyphenols to the large intestine, achieving high-concentration sustained delivery targeted to the colorectum.

[0031] (2) The porous starch-self-assembled polyphenol nanocarrier drug delivery system of the present application alleviates weight loss in individuals with colitis, alleviates colon shortening and colon damage caused by colitis, and reduces the level of intestinal inflammation in individuals with colitis.

[0032] (3) Compared with other existing polyphenol encapsulation methods, the porous starch-self-assembled polyphenol nanocarrier drug delivery system of the present application has the advantages of cheap and easy-to-obtain wall materials, wide sources, simple equipment required, simple preparation process, good biosafety, low energy consumption in the preparation process, and no generation of toxic and hazardous waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The dissolution curves of Qu, Qu-PS, OVA / CMC-Qu and Qu-Nano-PS in deionized water.

[0034] Figure 2 The dissolution curves of Qu, Qu-PS, OVA / CMC-Qu and Qu-Nano-PS in artificial gastric fluid.

[0035] Figure 3 These are the dissolution curves of Qu, Qu-PS, OVA / CMC-Qu and Qu-Nano-PS in artificial intestinal fluid.

[0036] Figure 4 The quercetin release curves of Qu, PS, Qu-PS, OVA / CMC-Qu and Qu-Nano-PS during simulated gastric, small intestine and large intestine digestion.

[0037] Figure 5 Effects of Qu, PS, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS on colon length in a DSS-induced colitis mouse model (image (A) and colon length (B)).

[0038] Figure 6 The effects of Qu, PS, Qu-PS, OVA / CMC-Qu and Qu-Nano-PS on the colon inflammation level in colitis mice; among them, A is the TNF-α level and B is the IL-6 level. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0041] Example 1:

[0042] 10g of commercially available porous starch (particle size approximately 10 microns, pore diameter approximately 1 micron) was accurately weighed and placed in a 50mL beaker. 50mL of a 10mg / mL quercetin ethanol solution was added and placed in a magnetic stirrer at 200rpm. After adsorption for 60 minutes, a portion of the sample was removed and centrifuged at 4000×g for 5 minutes. The sample was freeze-dried, ground, and passed through a 100-mesh sieve to prepare quercetin-porous starch microcapsules, designated Qu-Ps.

[0043] Example 2:

[0044] (1) 2% by weight of sodium carboxymethyl cellulose (CMC) was dissolved in ultrapure water and heated at 60°C for 0.5 h to obtain a CMC solution. OVA (ovalbumin) lyophilized powder was added to the CMC solution to achieve a final OVA concentration of 1%. The pH was adjusted to 2.0, and the solution was immediately heated in an 80°C water bath for 30 min to obtain an OVA / CMC nanoparticle solution.

[0045] (2) A 10 mg / mL quercetin-ethanol working solution was prepared, and then 3 mL of the quercetin-ethanol working solution was added to 50 mL of the OVA / CMC nanoparticle solution. The ethanol in the solution was removed using a rotary evaporator to obtain an OVA / CMC-Qu nanoparticle solution. A batch of OVA / CMC-Qu nanoparticle solutions was prepared under the same conditions and freeze-dried to obtain OVA / CMC-Qu nanoparticles, which were stored at -20°C as control samples.

[0046] (3) 5 g of porous starch (same as in Example 1) was dispersed in 50 mL of OVA / CMC-Qu nanoparticle solution (0.5 mg / mL). A magnetic stirrer was used to uniformly disperse the porous starch in the solution for adsorption for 15 min. The system was filtered using a filter paper with a pore size of 5 μm to obtain a filter residue. The filter residue was freeze-dried to prepare a porous starch-self-assembled quercetin nanocarrier loading system, recorded as Qu-Nano-PS, and stored at -20 °C.

[0047] Example 3 Determination of in vitro dissolution saturation solubility

[0048] The in vitro dissolution saturation solubility was determined as follows:

[0049] The final solution was made up to volume with deionized water, wherein every 1000 mL of artificial gastric fluid contained 3.84 mL of saturated concentrated hydrochloric acid, 5 mL of Tween-80, 100 mg of α-amylase and saccharifying enzyme (the mass ratio of α-amylase to saccharifying enzyme was 1:1), and the pH of the solution was maintained at 1.5.

[0050] The final solution was fixed to volume with deionized water, wherein each 1000 mL of artificial intestinal fluid contained 100 mg of α-amylase and saccharifying enzyme, 5 mL of Tween-80 and 6.8 g of dipotassium hydrogen phosphate, and the pH of the solution was maintained at 7.4.

[0051] Excess quercetin Qu, OVA / CMC-Qu (prepared in Example 2), Qu-Ps (prepared in Example 1) and Qu-Nano-PS (prepared in Example 2) were weighed and placed in 30 mL of deionized water, artificial gastric juice and artificial intestinal juice, respectively, so that the final concentration of quercetin in the system was 12.5 mg / mL. The entire solution was incubated at 37°C, 1 mL of sample was taken out at different time points, and the same volume of the same solution was added to the system. The precipitate was removed by centrifugation at 4000g for 10 minutes at 4°C, and the supernatant was extracted with the same volume of carbon tetrachloride. The quercetin concentration was measured at 373 nm using a microplate reader (the standard curve was y = 0.0435x + 0.0102, R 2 =0.9998), the experiment was repeated three times, and the average value was taken to obtain the saturated dissolution curves of quercetin microcapsules in deionized water, artificial gastric juice and artificial intestinal juice.

[0052] Figure 1 The dissolution curves of Qu, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS in deionized water are presented. The dissolution release of quercetin continued to increase between 0 and 6 hours. Between 6 and 48 hours, the quercetin content in deionized water tended to level off and slightly decreased. The cumulative release of Qu-PS in deionized water increased rapidly between 0 and 12 hours, then slowly increased between 12 and 24 hours, reaching equilibrium between 24 and 48 hours. The dissolution release of OVA / CMC-Qu increased rapidly in the first 12 hours, then the rate of increase slowed between 12 and 48 hours, indicating that OVA / CMC-Qu exhibited sustained-release characteristics in deionized water. Encapsulation of quercetin using porous starch or nanoparticles can improve its saturated solubility and stability in water to a certain extent. The dissolution release of Qu-Nano-PS increased rapidly between 0 and 12 hours and reached saturated solubility. Between 24 and 48 hours, the dissolution rate increased slightly. According to the saturation solubility test results, it can be seen that the use of porous starch self-assembled nanocarriers to encapsulate quercetin can improve the saturated solubility of quercetin in water and, to a certain extent, improve the stability of quercetin in water.

[0053] Figure 2 The dissolution profiles of Qu, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS in artificial gastric fluid are presented. Quercetin reached its highest solubility at 6 hours and subsequently reached equilibrium. The presence of Tween-80 increased the saturation solubility of quercetin. The dissolution and release of Qu-PS in artificial gastric fluid initially increased rapidly between 0 and 12 hours, then slowly increased between 12 and 18 hours, and plateaued between 18 and 48 hours. OVA / CMC-Qu significantly increased the saturation solubility of quercetin in artificial gastric fluid. This is presumably due to the acidic conditions in which OVA / CMC nanoparticles were prepared and the decomposition of OVA in artificial gastric fluid, resulting in the higher solubility and faster release rate of OVA / CMC-Qu in artificial gastric fluid. However, this explosive growth may lead to cytotoxicity, and quercetin is destroyed and loses its bioactivity in gastric fluid, necessitating a secondary loading of OVA / CMC-Qu.

[0054] The solubility of Qu-Nano-PS increased rapidly between 0 and 12 hours, then slowly increased between 12 and 18 hours, leveling off between 18 and 48 hours, resulting in low solubility in artificial gastric fluid. This is presumably due to partial blockage of the pore entrance by nanoparticles and the high concentration of nanoparticles within the pores, which reduced contact between artificial gastric fluid and the nanoparticles within the pores, resulting in less quercetin release and, consequently, lower saturation solubility of Qu-Nano-PS in artificial gastric fluid.

[0055] Encapsulating quercetin solely with porous starch increased its saturated solubility in artificial gastric fluid. Premature release in artificial gastric fluid would result in a loss of bioactivity, hindering its bioavailability. Encapsulating quercetin solely with nanoparticles significantly increased its saturated solubility in artificial gastric fluid. Encapsulating quercetin with porous starch self-assembled nanocarriers, however, reduced its dissolution in artificial gastric fluid to a certain extent and improved its stability in the fluid.

[0056] Figure 3The dissolution curves of Qu, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS in artificial intestinal fluid are presented. Because the solubility of quercetin Qu in alkaline solutions is greater than that in acidic environments, its saturated solubility in artificial intestinal fluid is higher than that in deionized water and artificial gastric fluid. The solubility of quercetin Qu in artificial intestinal fluid rapidly reaches saturation in around 0.5 h, and its solubility rises faster than that of other samples. It is speculated that the hydrogen bonding between quercetin and porous starch affects the dissolution and release of quercetin in artificial intestinal fluid. The dissolution of Qu-PS increases rapidly between 0 and 12 h, slowly increases between 12 and 18 h, and levels off between 18 and 48 h. The saturated solubility of quercetin Qu and Qu-PS in artificial intestinal fluid were 12.09 and 48.30 μg / mL, respectively. The encapsulation of quercetin in artificial intestinal fluid could increase the saturated solubility of quercetin in artificial intestinal fluid by 4.00 times. The encapsulation of quercetin in porous starch could improve the saturated solubility of quercetin in artificial intestine.

[0057] The solubility of OVA / CMC-Qu increased rapidly within the first 12 hours. However, due to aggregation after freeze-drying, the OVA / CMC-Qu exhibited a loose, flocculent structure, resulting in high surface tension in water, which affected its dispersibility in artificial intestinal fluid. Some quercetin degraded in the alkaline environment before being released, resulting in a low release amount, with a slight increase in release between 24 and 48 hours.

[0058] The solubility of Qu-Nano-PS increased rapidly between 0 and 12 hours, increased slowly between 12 and 18 hours, and increased slightly between 18 and 48 hours. This is because the pore entrances of the Qu-Nano-PS sample were partially blocked by nanoparticles, preventing intestinal fluid from entering the pores and coming into direct contact with the nanoparticles, reducing quercetin degradation. On the other hand, due to quercetin's ability to dissolve easily in alkaline solutions, quercetin was released into the artificial intestinal fluid in the form of nanoparticles, from the outside to the inside, greatly enhancing the saturated solubility of quercetin in artificial intestinal fluid. Encapsulation of quercetin with nanoparticles increased the saturated solubility of quercetin in artificial intestinal fluid by 1.91 times. Encapsulation of quercetin with porous starch self-assembled nanocarriers increased the saturated solubility of quercetin in artificial gastric fluid by 4.93 times.

[0059] The results of in vitro saturation solubility experiments showed that the use of porous starch to load quercetin increased the saturated solubility of quercetin in deionized water and artificial intestinal fluid to varying degrees, with the increase in deionized water being the most significant. However, the high saturated solubility of quercetin in artificial gastric fluid may cause quercetin to be destroyed and lose its activity during digestion. On the other hand, due to the strong hydrogen bonds between quercetin and the inner wall of the porous starch, the saturated solubility of quercetin in artificial intestinal fluid was not significantly improved.

[0060] The use of nanoparticles to load quercetin has resulted in varying degrees of improvement in the saturated solubility of quercetin in deionized water, artificial gastric juice, and artificial intestinal juice. However, since the loading system with protein as the wall material will degrade in gastric juice, the explosive release of quercetin in gastric juice may lead to quercetin degradation. The use of porous starches of different morphologies for secondary loading of nanoparticles suppressed the problem of large-scale release of quercetin in gastric juice and further improved the saturated solubility of quercetin in deionized water and artificial intestinal juice. Furthermore, the porous starch self-assembled nanocarriers exhibited the characteristics of sustained release over a long period of time, providing a theoretical basis for the construction of intestinal-targeted bioactive substance delivery carriers.

[0061] Example 4 In vitro digestion simulation

[0062] The in vitro digestion simulation assay was performed as follows:

[0063] (I) Preparation of simulated body fluids

[0064] Escherichia coli and Lactobacillus casei were cultured in Luria-Bertani medium and MRS broth, respectively. The culture medium was sterilized at 121°C for 30 min, and activated Escherichia coli and Lactobacillus casei were inoculated and cultured at 37°C for a certain period of time until the final number of Escherichia coli and Lactobacillus casei colonies in the solution was 10 8 -10 10 CFU / mL of Escherichia coli and lactic acid bacteria suspensions.

[0065] The human body's digestive juices contain a variety of elements. In order to restore the complex environment of the human body as much as possible, a series of digestive juices are precisely prepared for use. The following digestive juices are all made to volume using 1000mL. Each 1000mL of digestive juice contains the following substances:

[0066] (1) Gastric juice: 13 mL concentrated hydrochloric acid; 36 mL CaCl2·2H2O (22.2 g / L); 2 g BSA; 5 g pepsin; pH = 1.5;

[0067] (2) Duodenal fluid: 12.6 mL KCl (89.6 g / L); 18 mL CaCl2·2H2O (22.2 g / L); BSA 2 g; pancreatin 18 g; lipase 3 g; pH = 8.0;

[0068] (3) Bile: 136.6 mL NaHCO3 (84.7 g / L); 20 mL CaCl2·2H2O (22.2 g / L); BSA 3.6 g; porcine bile salt 5 g; pH = 7.0.

[0069] (II) In vitro digestion simulation

[0070] According to the internal environment of the human body, the reaction temperature was set at 37°C, and the digestion time in the stomach, small intestine and large intestine were 2, 2 and 4 hours respectively.

[0071] (1) Gastric stage: 10 mg of each quercetin-containing sample (Qu, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS) was accurately weighed, added to 6 mL of deionized water (pH 6.8) and 12 mL of simulated gastric fluid, and reacted for 2 h;

[0072] (2) Small intestine stage: 12 mL of simulated duodenal fluid and 6 mL of simulated bile were added to the mixture obtained in step 1, the pH was adjusted to 7.4, and the mixture was reacted for 2 h.

[0073] (3) Large intestine stage: 36 mL of Escherichia coli and lactic acid bacteria suspension was added to the mixture obtained in step 2, and the pH was adjusted to 8.4 and the mixture was reacted for 4 hours.

[0074] (Ⅲ) In vitro release rate of quercetin

[0075] In vitro release rate of quercetin: After completing the simulated in vitro digestion, the digestion fluid was centrifuged (20,000 g, 30 min, 4°C). The supernatant was extracted with the same volume of carbon tetrachloride and the quercetin content was analyzed at 373 nm using a microplate reader. The portion that was soluble in the digestion fluid was considered to be the portion that could be absorbed in the micelles. The in vitro release rate of quercetin (Quercetin released) was calculated using the following formula:

[0076] Quercetin released (%) = C Micelles / C Digesta ×100% (Formula 1)

[0077] Among them C Micelles and C Digesta are the concentration of quercetin dissolved in micelles and the total quercetin concentration in the digestive fluid, respectively.

[0078] Figure 4The release curves of quercetin from Qu, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS during simulated gastric, small intestinal, and large intestinal digestion are presented. In gastric juice, due to the low solubility of quercetin under acidic conditions and the fact that starch is not degraded in gastric juice, there was no significant difference in the release rate of all samples. Quercetin rapidly dissolved and released after contact with small intestinal fluid, reaching a maximum release rate after 30 minutes. Subsequently, due to the degradation of quercetin in the alkaline solution environment, the quercetin concentration gradually decreased. After Qu-PS entered the small intestine for simulated digestion, starch hydrolases degraded some of the porous starch, and the highest release rate was reached at 30 minutes. The liquid after digestion cannot be completely absorbed by the human body. The part that is soluble in the digestive fluid is considered to be the part that can be absorbed in the micelles. That is, the quercetin that exists in the form of micelles in the chymosus after digestion is considered to be bioavailable. After simulated digestion in the stomach and small intestine, quercetin in the form of micelles is believed to be able to enter the body through the small intestinal epithelial cells. Encapsulating quercetin with porous starch can improve its bioavailability, but due to the hydrogen bonds between quercetin and the porous starch, the dissolution and release of quercetin are hindered, so the improvement in quercetin's bioavailability is relatively limited.

[0079] After entering the simulated large intestine, quercetin dissolution increased within the first 10 minutes due to the increased pH. Subsequently, due to the alkaline environment and the degradation rate of large intestinal microorganisms exceeding the dissolution rate, the quercetin concentration continued to decrease. The porous starch loading significantly improved quercetin stability in the large intestine. Qu-PS reached a peak release rate of 50.66% during the large intestine. However, since the majority of quercetin in Qu-PS is distributed within the surface pores of the starch granules, it remains in full contact with digestive fluids, resulting in limited improvement in quercetin stability. The porous starch loading enhanced quercetin's bioavailability and digestive stability, resulting in high concentrations of quercetin entering the colorectum after digestion in the stomach and small intestine. However, hydrogen bonding between quercetin and the porous starch wall partially hindered its release. During gastric digestion, a high concentration of quercetin dissolved, leading to its partial degradation. This limits the improvement of quercetin bioavailability and the prolonged release of quercetin in the large intestine.

[0080] The release profile of OVA / CMC-Qu during simulated digestion was generally consistent with its in vitro dissolution pattern. The amphiphilic nature of the OVA / CMC nanoparticles significantly enhanced quercetin solubility. Furthermore, during gastric digestion, degradation of the protein-based carrier wall material led to leakage of the core material, resulting in higher gastric release of the OVA / CMC-Qu sample compared to the other samples. During simulated small intestinal digestion, OVA / CMC-Qu reached its peak quercetin release around 30 minutes into the digestive process. Quercetin, present in micellar form in digestive fluids after gastric and small intestinal digestion, is generally considered directly absorbable. Nanoparticle loading increased quercetin bioavailability by approximately 20-fold. During large intestinal digestion, the wall material degraded completely, resulting in the majority of quercetin being released in the stomach and small intestine, leaving only a small amount entering the large intestine. The concentration of quercetin continued to decrease due to pH and microbial degradation.

[0081] During the simulated digestion phases of the stomach and small intestine, hydrogen bonding between the quercetin in the Qu-Nano-PS nanoparticles and the porous starch inner wall enhances the stability of the porous starch self-assembled nanocarriers and increases their bioavailability by approximately 30-fold. The porous starch self-assembled nanocarriers reach a peak release rate of 93.69% upon entering the large intestine. Compared to quercetin directly adsorbed onto porous starch, the nanocarriers exhibit higher solubility in the large intestine. Compared to quercetin adsorbed onto nanoparticles, the nanocarriers enhance gastric digestion stability, avoid the biotoxicity associated with explosive release of quercetin in the stomach, and successfully deliver quercetin to the colorectum for utilization by the organism.

[0082] Encapsulating quercetin in the form of nanoparticles in porous starch channels reduces the generation of hydrogen bonds between porous starch and quercetin, avoiding the restriction of quercetin dissolution and release due to strong hydrogen bonding. On the other hand, the steric hindrance caused by the nanoparticles limits the direct contact between the digestive fluid and quercetin, thereby improving digestion stability and allowing higher quercetin concentrations to enter the colorectal area. Due to the special physical structure of Qu-Nano-PS, quercetin slowly disperses into the digestive fluid along with the nanoparticles, achieving continuous high-concentration quercetin release in the large intestine. Through porous starch self-assembled nanocarriers, it is expected that the total amount of drug administered during oral intake can be reduced while maintaining the same local drug concentration, reducing damage to biological cells due to drug leakage and high-concentration original drugs, and achieving the goal of "reducing toxicity and increasing efficacy."

[0083] Example 5 In vivo treatment of ulcerative colitis

[0084] The in vivo therapeutic effect of the porous starch-self-assembled quercetin nanocarrier loading system on ulcerative colitis was analyzed as follows:

[0085] Female C57BL / 6 mice (6 weeks, 18-20 g) were divided into 8 groups, 8 mice each. After 1 week of acclimatization, the mice were divided into normal group, model group, Qu group, OVA / CMC-Qu group, Qu-PS group, and Qu-Nano-PS group.

[0086] For the first two weeks, the normal and model groups drank only sterile distilled water. The remaining groups received a low-dose oral dose (40 mg / kg / day). From days 15 to 21, all groups, except the blank control group, received 3.5% DSS in their drinking water to induce colitis. After 21 days, the mice were euthanized, and the colons were photographed and their lengths recorded. ELISA kits were used to measure TNF-α and IL-6 concentrations in the rat colonic tissue.

[0087] Figure 5 The effects of Qu, PS, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS on colon length in a DSS-induced colitis mouse model. DSS causes colon atrophy in mice while inducing colitis, so colon length is often used as one of the criteria for evaluating the severity of colitis. Figure 4 As shown in (A) and (B), the average colon length in the DSS group was 5.1 cm, which was 75.00% of the average colon length in the normal group (the average colon length of mice in the normal group was 6.8 cm). Oral administration of Qu and Qu-PS to mice had a small effect on the shortening of colon length. Oral administration of OVA / CMC-Qu improved the shortening of colon length to a certain extent. Oral administration of Qu-Nano-PS significantly improved the shortening of colon length. The OVA / CMC-Qu group and the Qu-Nano-PS group improved to 6.1 cm and 6.6 cm respectively. The effect of oral administration of Qu-Nano-PS was significantly better than that of OVA / CMC-Qu.

[0088] Figure 6 The effects of Qu, PS, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS on colonic inflammation in colitis mice were investigated. Inflammatory cytokines play a crucial role in the development and progression of ulcerative colitis (UC). Therefore, to evaluate the effects of different carriers on UC, their effects on TNF-α and IL-6 production, both well-known markers of inflammation, were investigated.

[0089] TNF-α is an important cytokine that activates inflammatory cells and causes inflammatory responses, and is therefore generally considered a pro-inflammatory factor. Figure 6In (A), the TNF-α level in the colon of the model group mice was significantly higher than that in the normal group (from 9.77 pg / mg in the normal group to 54.63 pg / mg in the model group), but Qu, PS, Qu-PS, OVA / CMC-Qu and Qu-Nano-PS were able to reduce its level in the colon to varying degrees, which were 52.14 pg / mg, 45.52 pg / mg, 36.06 pg / mg and 27.32 pg / mg respectively. Among them, the TNF-α level of mice after Qu-Nano-PS intervention was reduced to 60.0% compared with that of the model group mice. Figure 6 In (B), Qu, PS, Qu-PS, OVA / CMC-Qu, and Qu-Nano-PS all reduced IL-6 levels in the colon to varying degrees, with Qu-Nano-PS intervention leading to the most significant reduction in IL-6 levels. This suggests that Qu-Nano-PS has the potential to effectively inhibit the progression of UC. The results showed that treatment with Qu-Nano-PS significantly reduced TNF-α ( Figure 6 A) and IL-6 ( Figure 6 These findings indicate that Qu-Nano-PS has the potential to effectively inhibit the progression of UC.

[0090] In summary, the present application will load the nanoparticles loaded with polyphenols into the porous starch pores as a whole, and construct a porous starch-self-assembled polyphenol nanocarrier loading system. Compared with the single loading system, the digestion stability and bioavailability of polyphenols are further improved. By performing in vitro simulated digestion analysis on porous starch self-assembled nanocarriers, it was found that this loading system can inhibit the dissolution, release and degradation of polyphenols in the simulated fluid of the stomach and small intestine, thereby maintaining a long-term high concentration of polyphenol release in the simulated digestion stage of the large intestine. It significantly improves the atrophy of the mouse colon caused by colitis, protects colon tissue, reduces the structural damage induced by DSS, and can ensure the sufficient concentration of polyphenols in the inflamed colon and prolong the residence time, thereby effectively reducing the inflammatory response. It is conducive to constructing a carrier for long-term, high-concentration, targeted colorectal release, and while maintaining the same drug release amount, reduces the amount of drug delivered, thereby avoiding the biological toxicity caused by high-concentration original drugs.

[0091] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. Use of porous starch-self-assembled nanocarrier microcapsules in the preparation of drugs for preventing or treating ulcerative colitis, characterized in that: The preparation of the porous starch-self-assembled nanocarrier microcapsules comprises: (1) adjusting the pH of a mixed aqueous solution containing sodium carboxymethyl cellulose (CMC) and ovalbumin (OVA) to acidic, and then heating the mixture in a water bath to react, thereby obtaining an OVA / CMC nanoparticle solution; (2) adding a short-chain alcohol solution of polyphenols to the obtained OVA / CMC nanoparticle solution to obtain a polyphenol mixed solution, and evaporating the short-chain alcohol in the polyphenol mixed solution to obtain an OVA / CMC-Qu nanoparticle solution; (3) The porous starch is uniformly dispersed in the aqueous solution of OVA / CMC-Qu nanoparticles for dynamic adsorption. After the adsorption is completed, the resulting reaction solution is filtered, and the filter residue is collected and dried.

2. The use according to claim 1, characterized in that In step (1): In the mixed aqueous solution, the mass percentage concentration of sodium carboxymethyl cellulose CMC is 1-3%, and the mass percentage concentration of ovalbumin OVA is 0.5-1.5%. The pH value is adjusted to 2-3. The water bath temperature is 70-80° C. and the reaction time in the water bath is 20-40 minutes.

3. The use according to claim 1, characterized in that In step (2): The polyphenol is at least one of quercetin, curcumin, and resveratrol; The short-chain alcohol is methanol, ethanol or propanol; The concentration of polyphenols in the short-chain alcohol solution of polyphenols is 5-15 mg / mL; the volume ratio of the short-chain alcohol solution of polyphenols to the OVA / CMC nanoparticle solution is 1-5:

50.

4. The use according to claim 1, characterized in that In step (3): The concentration of the OVA / CMC-Qu nanoparticle aqueous solution is 0.01 to 15 mg / mL; the mass volume ratio of the porous starch to the OVA / CMC-Qu nanoparticle aqueous solution is 3 to 10 g:50 mL; The dynamic adsorption is carried out under shaking or stirring conditions; the adsorption time is 10 to 20 minutes.

5. The use according to claim 1, characterized in that The drug is used for at least one of the following: Improve colon atrophy or colon damage caused by colitis; Inhibit the production of pro-inflammatory factors in colon tissue; Relieves weight loss in individuals with colitis.

6. The use according to claim 5, characterized in that The pro-inflammatory factors include TNF-α and IL-6.

7. The use according to claim 1, characterized in that The drug is an oral preparation, and the dosage form of the oral preparation is selected from tablets, liquid, powder or soft gel.

8. The use according to claim 1, characterized in that The medicine further contains pharmaceutically acceptable excipients.

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