Starch-indolecarboxylic acid derivatives, methods for producing the same, and their use

Abstract

The present application relates to the technical field of modified starch and provides a starch-indole carboxylic acid derivative and its preparation and use. The starch-indole carboxylic acid derivative refers to esterified starch produced by the esterification reaction of starch and indole carboxylic acid under the action of condensing agent and base, which has high resistance and can withstand the degradation in stomach and small intestine, and can release indole carboxylic acid beneficial to intestinal health through fermentation by intestinal flora after reaching the colon region, which has obvious advantages over traditional administration methods such as intragastric administration and intraperitoneal injection, and can significantly increase the content of indole carboxylic acid in colon and hepatic portal blood, and indole carboxylic acid can activate aryl hydrocarbon receptor to play an immunomodulatory role, and the regulatory role of short-chain fatty acid released by intestinal flora fermentation of starch is superimposed to provide a product that exerts a synergistic immunomodulatory role through multiple immune system signaling pathways.

Description

Technical Field

[0001] This application relates to the technical field of modified starch, and particularly to starch-indole carboxylic acid derivatives, their manufacturing methods, and uses.

[0002] <Cross-reference to Related Applications> This application claims the priority of a Chinese patent application with an application number of 202111213344.7 and an invention title of "Starch-Indole Carboxylic Acid Derivatives, Their Manufacturing Methods, and Uses", which was filed with the China National Intellectual Property Administration on October 19, 2021, and incorporates all of its content by reference.

Background Art

[0003] Indole carboxylic acid derivatives such as 3-indoleacetic acid (IAA), 3-indolepropionic acid (IPA), 3-indolelactic acid (ILA), and 3-indoleacrylic acid (IA) are metabolites of tryptophan by intestinal microorganisms and play important roles in regulating the balance of intestinal immunity. IAA, IA, ILA, etc. can regulate the balance of intestinal immunity as ligands of the aryl hydrocarbon receptor (AhR), and it has been found in research that IAA suppresses the levels of pro-inflammatory cytokines in mouse macrophages and hepatocytes in an AhR-dependent manner, and further reduces the inflammatory response of the liver. IPA, as a ligand of the pregnane X receptor (PXR), regulates the intestinal barrier function of mice, especially in the presence of indole, and an increase in the serum IPA concentration is associated with a decrease in the incidence of type 2 diabetes, insulin secretion, and insulin resistance. Also, IPA can act as a hydroxyl radical scavenger to exhibit antioxidant effects.

[0004] Tryptophan is an essential amino acid for humans and is supplied from food proteins. The metabolism of tryptophan in the digestive tract mainly involves three pathways: the kynurenine pathway, the 5-hydroxytryptamine pathway, and direct degradation by gut microbiota. Gut microbiota are most abundant in the colon, the main site where they break down tryptophan to produce indole carboxylic acids such as IAA / IPA. Studies have shown that IAA content in the feces of obese and diabetic patients is significantly lower than in healthy individuals, and epidemiological studies have shown a negative correlation between serum IPA levels and type 2 diabetes (T2D) and mild inflammation, suggesting that the content of indole carboxylic acids such as IAA / IPA is closely related to human diseases. Therefore, targeted delivery of indole carboxylic acids such as IAA / IPA to the colon is expected to have preventive or therapeutic effects on inflammatory and autoimmune diseases such as inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease, and multiple sclerosis, by utilizing the beneficial immunomodulatory effects of indole carboxylic acids such as IAA / IPA.

[0005] However, when these small molecules of indolecarboxylic acid are ingested orally, they are absorbed in the stomach or small intestine of the digestive tract without actually reaching the colon. How to deliver these immunomodulatory indolecarboxylic acid molecules to the target colon site and exert their effects remains a challenge to be addressed.

[0006] This application was made in light of the circumstances described above. [Overview of the project] [Problems that the invention aims to solve]

[0007] The first object of this application is to provide a starch-indolecarboxylic acid derivative that can withstand degradation in the stomach and small intestine, and after reaching the colon, can release indolecarboxylic acid through fermentation by the intestinal flora, thus having clear advantages compared to conventional methods of administering indolecarboxylic acid such as intragastric administration and intraperitoneal injection, and can significantly increase the indolecarboxylic acid content in the colon and hepatic portal blood.

[0008] A second object of this application is to provide a method for producing starch-indole carboxylic acid derivatives.

[0009] A third object of the present invention is to provide the use of starch-indole carboxylic acid derivatives, which can be broadly used in the manufacturing process of products that modulate the balance of intestinal immunity. [Means for solving the problem]

[0010] In order to achieve the above-mentioned objectives of this application, the following technical means will be employed in particular. The starch-indolecarboxylic acid derivative provided by this application is mainly produced by an esterification reaction of starch and indolecarboxylic acid using a condensing agent and a base. Preferably, after condensation, the degree of substitution of the indolecarboxylic acid in the starch-indolecarboxylic acid derivative is 0.01 to 1.0.

[0011] Furthermore, the indolecarboxylic acid comprises at least one of indoleacetic acid, indolepropionic acid, indoleacrylic acid, or indolelactic acid.

[0012] Furthermore, the starch includes at least one of high-amylose corn starch (HAMS), potato starch, sweet potato starch, mixed bean starch, and banana starch.

[0013] Furthermore, the condensing agent comprises at least one of EDCI, DCC, and HATU. Preferably, the base comprises at least one of 1-methylimidazole, an amine, sodium bicarbonate, and sodium carbonate.

[0014] A method for producing the above-mentioned starch-indolecarboxylic acid derivative provided by this application, wherein the production method is: (a) Dissolve starch in a solvent to obtain solution A, then add indolecarboxylic acid, a condensing agent and a base to solution A to carry out an esterification reaction to obtain reaction solution A, (b) The step of precipitating starch in reaction solution A, followed by suction filtration and drying in that order to obtain a starch-indole carboxylic acid derivative.

[0015] Furthermore, the solvent in step (a) includes at least one of DMSO, an ionic liquid, and water.

[0016] Furthermore, the temperature of the esterification reaction in step (a) is 20-80°C, and the duration is 20-30 hours.

[0017] Furthermore, the precipitation method in step (b) involves adding reaction solution A dropwise to ethanol or water to precipitate the starch-indole carboxylic acid derivative.

[0018] The use of the above-mentioned starch-indolecarboxylic acid derivative provided herein in the manufacture of a product that modulates the balance of intestinal immunity.

[0019] The aforementioned regulation of the balance of intestinal immunity refers to the fact that, after the starch-indole carboxylic acid derivative is ingested and reaches the colon, the linked indole carboxylic acid is released through fermentation by the intestinal microbiota. This synergistically interacts with short-chain fatty acids released by the intestinal microbiota fermentation of starch, exerting an immunomodulatory effect through multiple immune system signaling pathways.

[0020] Furthermore, the products that regulate the balance of the intestinal immunity include drugs, health foods, foods for specified health uses, or general foods.

[0021] Furthermore, the drug is a drug for preventing and treating inflammatory diseases and / or autoimmune diseases.

[0022] Furthermore, the inflammatory disease and / or autoimmune disease includes one of inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease, and multiple sclerosis.

Advantages of the Invention

[0023] This application has the following beneficial effects compared with the prior art. As the starch-indolecarboxylic acid derivative provided by this application, the starch-indolecarboxylic acid derivative is mainly produced by the esterification reaction of starch and indolecarboxylic acid under the action of a condensing agent and a base. The above starch-indolecarboxylic acid derivative is an acylated starch formed by the esterification reaction of starch and indolecarboxylic acid, has high resistance, can withstand the decomposition in the stomach and small intestine, and can release indolecarboxylic acid through the fermentation of the intestinal flora after reaching the colon site. It has obvious advantages compared with the conventional administration methods of indolecarboxylic acid such as oral administration and intraperitoneal injection in the stomach, and can significantly increase the content of indolecarboxylic acid in the colon and portal vein blood.

[0024] The indolecarboxylic acid released from the starch-indolecarboxylic acid derivative provided by this application at the colon site exhibits an immunomodulatory effect by activating AhR. In addition, the short-chain fatty acids generated by the fermentation of starch by the intestinal flora can also exert an immunomodulatory effect by activating G protein-coupled receptors and inhibiting histone deacetylases. By positively superimposing the effects of indolecarboxylic acid and short-chain fatty acids, it is possible to provide a product that synergistically exhibits an immunomodulatory effect through multiple immune system signal transduction pathways.

[0025] The method for producing a starch-indolecarboxylic acid derivative provided by this application involves dissolving starch in a solvent to obtain solution A, then adding indolecarboxylic acid, a condensing agent, and a base to solution A to carry out an esterification reaction to obtain reaction solution A, and then precipitating the starch in reaction solution A, followed by suction filtration and drying in that order to obtain the starch-indolecarboxylic acid derivative. The above production method has the advantage of being simple in its processing steps and suitable for industrial mass production.

[0026] The starch-indolecarboxylic acid derivatives provided by this application can be widely used in products that regulate the balance of intestinal immunity and in products that prevent and treat inflammatory diseases. [Brief explanation of the drawing]

[0027] In order to more clearly describe the specific embodiments of the present application or the technical means in the prior art, the drawings used in the description of the specific embodiments or the prior art are briefly introduced below. Needless to say, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings from these drawings without performing any novel work.

[0028] [Figure 1] Figure 1 shows the hydrogen nuclear magnetic resonance spectrum of the starch-indole carboxylic acid derivative provided by Example 1 of the present application. [Figure 2] Figure 2 shows the infrared spectra of starch (HAMS) and indoleacetic acid-derivative high-amylose corn starch (HAMSIAA) provided by Example 1 of the present application. [Figure 3] Figure 3 shows the XRD patterns of starch (HAMS) and indoleacetic acid-derived high-amylose corn starch (HAMSIAA) provided by Example 1 of the present application. [Figure 4]Figure 4 shows SEM electron microscope images of starch (HAMS) and indoleacetic acid derivative high-amylose corn starch (HAMSIAA) provided by Example 1 of the present application. [Figure 5] Figure 5 shows the concentration of indolecarboxylic acid in the feces of experimental mice provided by Example 2 of the present application. [Figure 6] Figure 6 shows the concentration of indolecarboxylic acid in the hepatic portal blood of experimental mice provided by Example 2 of the present invention. [Figure 7] Figure 7 shows the concentration of indolecarboxylic acid in the feces of experimental mice provided by Example 3 of the present application. [Figure 8] Figure 8 shows the concentrations of IAA in colon contents for different administration methods provided by Example 4 of the present application. [Figure 9] Figure 9 shows the concentrations of IAA in hepatic portal blood serum for different administration methods provided by Example 4 of the present application. [Figure 10] Figure 10 shows the concentration of IPA in the feces of experimental mice provided by Example 5 of the present application. [Figure 11] Figure 11 shows a comparison of IAA / IPA concentrations in peripheral blood serum between a normal group and a group with DSS-induced ulcerative colitis, as provided by Example 6 of the present application. [Figure 12] Figure 12 shows the change in body weight of mice after treatment with 15% HAMSIAA-0.49 provided by Example 6 of the present application. [Figure 13] Figure 13 shows the change in colon length of mice after treatment with 15% HAMSIAA-0.49 provided by Example 6 of the present application. [Figure 14] Figure 14 shows the change in the DAI index of mice after treatment with 15% HAMSIAA-0.49 provided by Example 6 of the present application. [Figure 15] Figure 15 shows the changes in the colonic condition of mice after treatment with 15% HAMSIAA-0.49 provided by Example 6 of the present application. [Figure 16]Figure 16 shows the changes in a mouse colon tissue section after treatment with 15% HAMSIAA-0.49 provided by Example 6 of the present application. [Figure 17] Figure 17 shows the content of the anti-inflammatory cytokine IL-10 in the colon tissue of a mouse provided by Example 6 of the present application. [Figure 18] Figure 18 shows the content of the pro-inflammatory cytokine IL-6 in the colon tissue of a mouse provided by Example 6 of the present application. [Figure 19] Figure 19 shows the content of the pro-inflammatory cytokine IL-1β in the colon tissue of a mouse provided by Example 6 of the present application. [Figure 20] Figure 20 shows the relative expression levels of AhR in mouse colon tissue provided by Example 6 of the present application. [Figure 21] Figure 21 shows the content of IL-22, an anti-inflammatory cytokine, in the colon tissue of a mouse provided by Example 6 of the present application. [Figure 22] Figure 22 shows comparative images of HE staining of kidney tissue from each group of mice provided in Example 7 of the present application. [Figure 23] Figure 23 shows the pathological scores of the kidney tissue of each group of mice provided by Example 7 of the present application. [Modes for carrying out the invention]

[0029] The technical means of the present application will be clearly and completely described below using examples, and it goes without saying that the examples described are only some, not all, examples of the present application. If a person skilled in the art obtains other examples based on the examples of the present application without performing any novel work, all of them will fall within the scope of the protection of the present application.

[0030] According to one aspect of the present application, a starch-indolecarboxylic acid derivative is produced mainly by the esterification reaction of amylose starch and indolecarboxylic acid using a condensing agent and a base. Preferably, after condensation, the degree of substitution of the indolecarboxylic acid in the starch-indolecarboxylic acid derivative is 0.01 to 1.0.

[0031] The starch-indolecarboxylic acid derivative provided by this application is mainly produced by the esterification reaction of starch and indolecarboxylic acid using a condensing agent and a base. The above-mentioned starch-indolecarboxylic acid derivative is an acylated starch formed by the esterification reaction of starch and indolecarboxylic acid, possesses high resistance, can withstand degradation in the stomach and small intestine, and can release indolecarboxylic acid after reaching the colon through fermentation by the intestinal flora. It has clear advantages compared to conventional methods of administering indolecarboxylic acid such as intragastric administration and intraperitoneal injection, and can significantly increase the indolecarboxylic acid content in the colon and hepatic portal blood.

[0032] Furthermore, by targeting the colon and releasing indolecarboxylic acid, inflammatory and autoimmune diseases can be effectively prevented and treated. However, inflammatory and autoimmune diseases include, but are not limited to, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease, and multiple sclerosis.

[0033] Preferably, the starch-indole carboxylic acid derivative comprises indole carboxylic acid derivatized starch and is a mixture of one or more of indole acetylated starch, indole propionylated starch, indole lactylated starch, and indole acrylicated starch.

[0034] In preferred embodiments of the present application, the indolecarboxylic acid comprises at least one of indoleacetic acid, indolepropionic acid, indoleacrylic acid, or indolelactic acid.

[0035] In preferred embodiments of the present application, the starch comprises at least one of high-amylose corn starch (HAMS), potato starch, sweet potato starch, mixed bean starch, and banana starch.

[0036] Furthermore, the term "high-amylose starch" refers to starch in which the amylose starch content exceeds 50%.

[0037] In a preferred embodiment of the present application, the condensing agent comprises at least one of EDCI, DCC, and HATU. In preferred embodiments of the present application, the base comprises at least one of 1-methylimidazole, an amine, sodium bicarbonate, and sodium carbonate.

[0038] According to one aspect of the present application, a method for producing the above-mentioned starch-indolecarboxylic acid derivative, wherein the production method is: (a) Dissolve starch in a solvent to obtain solution A, then add indolecarboxylic acid, a condensing agent and a base to solution A to carry out an esterification reaction to obtain reaction solution A, (b) The step of precipitating starch in reaction solution A, followed by suction filtration and drying in that order to obtain a starch-indole carboxylic acid derivative.

[0039] The method for producing a starch-indolecarboxylic acid derivative provided by this application involves dissolving starch in a solvent to obtain solution A, then adding indolecarboxylic acid, a condensing agent, and a base to solution A to carry out an esterification reaction to obtain reaction solution A, and then precipitating the starch in reaction solution A, followed by suction filtration and drying in that order to obtain the starch-indolecarboxylic acid derivative. The above production method has the advantage of being simple in its processing steps and suitable for industrial mass production.

[0040] In a preferred embodiment of the present application, the solvent in step (a) comprises at least one of DMSO, an ionic liquid, and water.

[0041] In a preferred embodiment of the present invention, the temperature of the esterification reaction in step (a) is 20 to 80°C, and the duration is 20 to 30 hours.

[0042] In a preferred embodiment of the present invention, the precipitation method in step (b) is to add reaction solution A dropwise to ethanol or water to precipitate the starch-indole carboxylic acid derivative.

[0043] According to one aspect of the present invention, the above-mentioned starch-indole carboxylic acid derivative is used in the manufacture of a product that modulates the balance of intestinal immunity.

[0044] The starch-indolecarboxylic acid derivatives provided herein can be widely used in the manufacturing process of products that regulate the balance of intestinal immunity.

[0045] In preferred embodiments of the present invention, regulating the balance of intestinal immunity refers to delivering indolecarboxylic acid to the colon using a starch-indolecarboxylic acid derivative as a targeting vector.

[0046] Preferably, the product that regulates the balance of intestinal immunity includes drugs, health foods, foods for special medical purposes, or general foods.

[0047] The technical means of the present application will be further explained below using examples.

[0048] Example 1: Production of starch-indolecarboxylic acid derivatives 1. Production of indoleacetic acid derivative high-amylose corn starch (HAMSIAA) HAMS was added to DMSO and stirred until the solution clarified. IAA, EDCI, and 1-methylimidazole were added in that order, and stirring continued for 24 hours after the preparation was complete. After the reaction was complete, the reaction mixture was added dropwise to EtOH or H2O to precipitate the solid, filtered by suction, washed with EtOH or H2O, and dried.

[0049] 2. Production of indolepropionic acid derivatized high-amylose corn starch (HAMSIPA) HAMS was added to DMSO and stirred until the solution clarified. IPA, EDCI, and 1-methylimidazole were added in that order, and stirring continued for 24 hours after the preparation was complete. After the reaction was complete, the reaction mixture was added dropwise to EtOH or H2O to precipitate the solid, filtered by suction, washed with EtOH or H2O, and dried.

[0050] By adjusting the mixing ratio, HAMSIAA and HAMSIPA with different degrees of substitution (0.01 to 1.0) were obtained. HAMSIAA is used as an example (Table 1).

[0051] [Table 1]

[0052] 3. Measurement of the degree of substitution of indolecarboxylic acid-derivatized high-amylose corn starch Measuring the degree of substitution of indolecarboxylic acid-derivativeized high-amylose corn starch by titration involves two steps: basic hydrolysis of the ester bond and neutralization of the excess base. The specific procedure was as follows:

[0053] 0.2 g of starch was weighed and added to 10 mL of acetone and 1 mL of water. The mixture was sealed and magnetically stirred until homogeneous. 3 mL of NaOH aqueous solution (1 mol / L) was added and magnetically stirred for 30 minutes. 10 mL of 60°C warm water was added to wash the walls, and stirring continued for 2 minutes. After cooling, 3 drops of phenolphthalein reagent were added, and the solution was titrated with HCl (0.5 mol / L) until it became colorless. The solution was then allowed to stand at room temperature for 2 minutes until the color disappeared, and the volume of HCl was recorded. Three parallel measurements were performed.

[0054] The mass fraction ω of the indoleacetyl group in the indolecarboxylic acid derivatized high-amylose corn starch HAMSIAA was as follows:

[0055]

number

[0056] In the formula, V2 and V1 are the volumes (mL) of HCl used when titrating HAMS and HAMSIAA, respectively; c is the concentration of HCl (mol / L); m is the mass (g) of the HAMSIAA sample; and 158 is the molecular weight of the indoleacetyl group.

[0057] The degree of substitution DS was calculated based on the indoleacetyl group content as follows.

[0058]

number

[0059]

number

[0060] In the formula, 158 is the relative molecular mass of the indoleacetyl group, 162 is the relative molecular mass of each glucose unit of starch, and 1 is the relative atomic mass of the H atom.

[0061] Similarly, the degree of substitution for HAMSIPA was as follows:

[0062]

number

[0063]

number

[0064] In the formula, ω is the mass fraction of the indolepropionyl group in modified starch HAMSIPA, V2 and V1 are the volumes (mL) of HCl used when titrating HAMS and HAMSIPA, respectively, c is the concentration of HCl (mol / L), m is the mass (g) of the HAMSIPA sample, 172 is the relative molecular weight of the indolepropionyl group, and 162 is the relative molecular weight of each glucose unit in the starch.

[0065] 4. Characterization of the structure of indolecarboxylic acid derivatized starch Hydrogen nuclear magnetic resonance spectrum ( 1 The specific structural information was characterized using 1H NMR and infrared spectroscopy (FTIR), the crystallinity of the modified starch was measured by X-ray diffraction (XRD), and the ultrafine morphology of the starch granules was analyzed using a scanning electron microscope (SEM).

[0066] Figure 1 shows the hydrogen nuclear magnetic resonance spectra of the starch-indole carboxylic acid derivatives provided by this embodiment. As can be seen from Figure 1, compared to HAMS, HAMSIAA and HAMSIPA show a characteristic NH peak at a chemical shift of 10.83 ppm, and HAMSIPA also shows two new peaks at 2.96 ppm and 2.69 ppm, which are characteristic peaks of the two methylene groups in IPA.

[0067] Figure 2 shows the infrared spectra of starch (HAMS) and indoleacetic acid-derived high-amylose corn starch (HAMSIAA) provided by this example. As can be seen from Figure 2, at 400 cm⁻¹ -1 The peaks at 2930 cm² are stretching vibration peaks of OH or NH. The OH peak was broad, but the NH peak was sharp. This is because IAA contains NH, and as the degree of substitution increases, the sharp peak becomes increasingly apparent. -1 The peak observed was the stretching oscillation peak of CH. Compared to HAMS, HAMSIAA was 1728 cm. -1 Then, the stretching vibration peak of the carbonyl group appears as a new peak at 1728 cm⁻¹. -1 The carbonyl group peak at 745 cm² became stronger as the degree of substitution increased. As can be seen from the spectrum, -1 The peak at 1728cm² also intensified as the degree of substitution of HAMSIAA increased, and this peak was the out-of-plane bending vibration peak of the aromatic ring CH, i.e., the out-of-plane bending vibration peak of the indole ring CH of IAA. -1 The characteristic peak of the carbonyl group at 745 cm and -1 The appearance of a characteristic peak of the aromatic ring CH at this stage indicated that the esterification reaction of HAMS and IAA was successful.

[0068] Note: In Figure 2, DS represents the degree of substitution.

[0069] According to literature reports, starch granules are polycrystalline, and their crystalline structure differs depending on the plant species from which they originate, resulting in three main types of X-ray diffraction patterns (Type A, Type B, and Type C). Furthermore, Type V structures can be obtained through special methods such as starch acylation, and some genetically bred starches exhibited Type A+V, Type B+V, and Type C+V. Different crystalline forms of starch showed distinct characteristic peaks; Type A had strong peaks at 15°, 17°, 18°, and 23°; Type B had strong peaks at 5.6°, 17°, 22°, and 24°; Type C showed a combination of Type A and Type B, with a peak appearing at 5.6° compared to Type A and a strong peak appearing at 23° compared to Type B; and the characteristic peaks for Type V were at 12.5° and 19.5°.

[0070] Figure 3 shows the XRD patterns of starch (HAMS) and indoleacetic acid derivatized high-amylose corn starch (HAMSIAA) provided by this embodiment. As can be seen from Figure 3, HAMS showed peaks at 5.6°, 17°, 19.5°, and 22°, indicating a B-type crystalline structure, while HAMSIAA showed characteristic peaks at 12.5° and 19.5°, indicating a V-type structure, and the peaks became weaker as the degree of substitution increased. These results indicate that the crystalline structure of HAMS was destroyed by acylation.

[0071] Figure 4 shows SEM electron microscope images of starch (HAMS) and indoleacetic acid derivatized high-amylose corn starch (HAMSIAA) provided by this example. As can be seen from Figure 4, HAMS starch granules are mostly circular or oval in shape and have a smooth, crack-free surface. However, HAMSIAA starch granules after acylation with IAA have a rough and irregular surface. This is because the morphology of the starch granules was destroyed during the reaction process, when the product was first dissolved in DMSO and then precipitated by adding ethanol or water after the reaction was complete.

[0072] Example 2: Evaluation of the effect of targeting IAA delivery to the colon using indoleacetic acid derivative starch (HAMSIAA) Mouse feeds were prepared using HAMS and HAMSIAA with different degrees of substitution, each at a 15% additive rate. Eight-week-old mice were randomly divided into six groups of five based on body weight. The groups were either the HAMS group or the HAMSIAA group (six groups in total, with DS values ​​of 0.065, 0.12, 0.19, 0.29, 0.37, and 0.49, respectively). Feces and serum were collected from the mice.

[0073] Method for measuring IAA / IPA content in mouse feces, hepatic portal blood, peripheral blood, and colon tissue: The content of the metabolites IAA / IPA in each sample was detected using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The IAA / IPA content in mouse feces was measured as follows: Methanol was added to the collected feces, followed by vortex extraction, sonication, and centrifugation. The supernatant was taken to obtain an extract, which was then processed and calibrated with IAA / IPA internal standards (IAA-d5 / IPA-d2). The extract was then purified using an HLB solid-phase extraction cartridge (pre-treated with 5 mL of methanol and 5 mL of water respectively before use, then eluted and discarded with 5 mL of methanol / water mixture and 5 mL of water, and finally eluted with 5 mL of methanol). The eluent was subjected to HPLC-MS / MS analysis to detect IAA / IPA. The IAA / IPA content in the hepatic portal vein and peripheral blood was measured as follows. The collected whole blood was allowed to stand, then centrifuged at low temperature, and the supernatant was removed to obtain serum. Subsequently, using the same method as above, an internal standard substance was added to the serum, and it was purified using a solid-phase extraction column. The metabolite IAA / IPA in the eluent was detected using HPLC-MS / MS. The IAA / IPA content in the colon tissue was measured as follows: Methanol was added, and after sonication, the sample was polished uniformly for one layer. Then, after vortexing, sonication extraction, and centrifugation, the supernatant was removed to obtain an extract. Subsequently, using the same method as above, an internal standard substance was added to the serum, and it was purified using a solid-phase extraction column. The concentration of IAA / IPA in the extract was detected using HPLC-MS / MS.

[0074] Figure 5 shows the concentration of indole carboxylic acid in the feces of the experimental mice in this example. As can be seen from Figure 5, when the amount of modified starch added to the feed was 15%, the concentration of IAA in the feces was significantly higher in the HAMSIAA group compared to the HAMS group. However, when the degree of substitution of HAMSIAA was 0.37, the concentration of IAA in the feces was highest at approximately 5470 μmol / kg, which was about 1370 times higher than in the HAMS group (approximately 4 μmol / kg). This indicates that the retention capacity of IAA is related to the degree of substitution, and when the degree of substitution is low, the concentration of IAA in the feces increases as the degree of substitution increases, and when the degree of substitution reaches a certain value and increases further, the concentration of IAA actually decreases.

[0075] Figure 6 shows the concentration of indole carboxylic acid in the hepatic portal blood of the experimental mice in this example. As can be seen from Figure 6, when the amount of modified starch added to the feed was 15%, the concentration of IAA in the hepatic portal blood of mice was significantly higher in the HAMSIAA group compared to the HAMS group. The concentration of IAA in the hepatic portal blood of the HAMSIAA groups with different degrees of substitution differed somewhat from the rule for the change in the concentration of IAA in feces. However, the HAMSIAA group with a degree of substitution of 0.19 had the highest concentration of IAA in the hepatic portal blood, at approximately 2700 nmol / mL, which was about 2700 times higher than the HAMS group (approximately 1 nmol / mL). The HAMSIAA group with a degree of substitution of 0.37 had the highest concentration of IAA in feces. This may be because HAMSIAA with a degree of substitution of 0.37 has higher tolerance, and less was absorbed in the stomach or small intestine, while more reached the colon, where it was broken down and absorbed.

[0076] By detecting the concentration of IAA in mouse feces and hepatic portal blood, it was found that when the amount of modified starch added to the feed was 15%, the HAMSIAA group showed a significantly higher concentration of IAA compared to the HAMS group. This indicates that HAMSIAA can effectively deliver IAA to its target and release it slowly into the colon. However, HAMSIAA with a substitution degree of 0.37 showed the best effect in targeted delivery.

[0077] Example 3: Evaluation of the effect of targeting IAA delivery to the colon using indoleacetic acid derivative starch (HAMSIAA) Mouse feed was prepared using HAMS and HAMSIAA with different degrees of substitution, each at a 1.5% additive concentration. Eight-week-old mice were randomly divided into six groups based on body weight, with five mice in each group. The groups were either the HAMS group or the HAMSIAA group (six groups in total, with DS values ​​of 0.065, 0.12, 0.19, 0.29, 0.37, and 0.49, respectively). Feces and serum were collected from the mice.

[0078] Figure 7 shows the concentration of indole carboxylic acid in the feces of the experimental mice in this example. As can be seen from Figure 7, when the amount of modified starch added to the feed was reduced to 1.5%, the concentration of IAA in the feces was still significantly higher in the HAMSIAA group compared to the HAMS group, and the concentration of IAA in the feces of the HAMSIAA group, which had a substitution degree of 0.37, was the highest at about 860 μmol / kg, which was about 200 times higher than that of the HAMS group (about 4 μmol / kg).

[0079] Example 4: Evaluation of the effectiveness of targeted delivery of IAA by different administration methods The experiment involved dividing the participants into a total of five groups. 1. Blank control group 2. HAMSIAA-0.32 (1.5%) group (Each mouse consumed 3g of rat food daily, ingesting a total of 0.068 mmol of IAA). 3. IAANA drinking water group (IAANa concentration was 2.68 mg / mL, and each mouse drank 5 mL of water daily, ingesting a total of 0.068 mmol of IAANA). 4. Intragastric administration group (IAANa concentration was 67 mg / mL, and 0.2 mL was administered intragastricly daily, for a total intake of 0.068 mmol of IAANA) 5. Intraperitoneal injection group (IANA concentration was 67 mg / mL, with 0.2 mL injected daily, for a total intake of 0.068 mmol of IAANA)

[0080] The effects of targeting IAA delivery to the colon by different administration methods were evaluated by comparing the concentrations of IAA in the colon contents and hepatic portal blood of mice administered using four methods: intragastric administration, intraperitoneal injection, direct ingestion of IAA saline solution, and HAMSIAA. The results are shown in Figure 8. Figure 8 shows the concentrations of IAA in colon contents under different administration methods.

[0081] As can be seen from Figure 8, compared to the three administration methods of intragastric administration, intraperitoneal injection, and direct ingestion of IAA saline solution, HAMSIAA can deliver IAA to the colon more effectively. When the intake of IAA was equivalent, the concentration of IAA in the feces was significantly higher in the HAMSIAA group, about 200 times higher than in the control group. The concentrations of IAA in the feces of the intragastric administration group and the intraperitoneal injection group were equivalent, about 7 times higher than in the control group, and the concentration of IAA in the feces of the IAA drinking water group was about 30 times higher than in the control group. These experimental results show that, whether administered intragastricly or directly ingested with IAA saline solution, most of the IAA is absorbed in the stomach or small intestine, and only a small portion reaches the colon. However, HAMSIAA utilizes resistant starch to withstand the digestive action of the stomach and small intestine, reach the colon, and slowly release IAA through fermentation by intestinal microorganisms in the colon. By detecting the concentration of IPA in the colon contents of mice administered using different methods, it was found that the IPA concentrations in the intragastric, intraperitoneal injection, and IAAna drinking groups did not show any significant changes compared to the control group, but the IPA concentration in the HAMSIAA group was significantly increased compared to the control group.

[0082] Figure 9 shows the concentrations of IAA in hepatic portal blood serum under different administration methods. As can be seen from Figure 9, when the intake of IAA was equivalent, the concentration of IAA in hepatic portal blood of mice under different administration methods was significantly increased compared to the control group. However, there was no significant difference in serum IAA concentration among the intragastric administration group, the intraperitoneal injection group, and the IAA drinking water group. Nevertheless, the 1.5% HAMSIAA-0.32 group showed a serum IAA concentration approximately 10 times higher than the other administration methods. This suggests that the targeted delivery and absorption effect of HAMSIAA is significantly superior to other administration methods.

[0083] Example 5: Evaluation of the effect of targeting IPA delivery to the colon using indolepropionic acid derivative starch (HAMSIPA) Indole carboxylic acid derivatized starch with different degrees of substitution was added to mouse feed at ratios of 15% and 1.5%, respectively. Mice were fed the specially prepared feed for one week, and feces were collected to detect the IPA content. The results are shown in Figure 10. Figure 10 shows the concentration of IPA in the feces of experimental mice provided by this embodiment.

[0084] As can be seen in Figure 10, the concentration of IPA in feces was significantly higher in the HAMSIPA group compared to the HAMS group, and this was dependent on the degree of substitution and dose, with higher amounts added resulting in higher IPA concentrations in feces. However, the HAMSIPA group with a degree of substitution of 0.25 had even higher IPA concentrations in feces. This suggests that HAMSIPA also possesses good colon-targeted delivery capabilities for IPA.

[0085] Example 6: Reduction of dextran sulfate sodium (DSS)-induced acute ulcerative colitis by HAMSIAA Rat food was prepared using manufactured HAMSIAA at a predetermined additive ratio. Mice were then divided into four groups: a control rat food group, a HAMS group, a control rat food + DSS group, a HAMS + DSS group, and a HAMSIAA + DSS group with different substitution degrees. After the start of the experiment, changes in fecal morphology, mental state, and body weight of the mice were observed and recorded daily. Regular drinking water and DSS aqueous solution were changed once every three days, and on the seventh day after the start of the experiment, the DSS aqueous solution was replaced with regular drinking water.

[0086] After the experiment was completed, the disease active index (DAI) of the mice was evaluated, the length of the mouse colon was measured and the colon condition of the mice was evaluated from the sections, and the levels of cytokines such as IL-10, IL-6, and IL-22 in the mouse colon tissue were detected. Finally, the preventive and therapeutic effects of HAMSIAA on DSS-induced acute ulcerative colitis in mice were evaluated using these indicators.

[0087] Figure 11 shows a comparison of IAA / IPA concentrations in peripheral blood serum between the normal group and the DSS-induced ulcerative colitis group. As can be seen from Figure 11, the serum concentration of IAA / IPA was significantly lower in the control + DSS group compared to the control group, suggesting that IAA / IPA, a metabolite of tryptophan produced by intestinal microorganisms, is related to ulcerative colitis. Therefore, we hypothesize that supplementing with IAA / IPA reduces the severity of DSS-induced ulcerative colitis in mice.

[0088] Furthermore, using 15% HAMSIAA-0.49 as an example, this application demonstrates that adding HAMSIAA to the feed can effectively reduce the severity of DSS-induced mouse ulcerative colitis, based on indicators such as changes in mouse body weight, colon length, DAI index, colon condition, and colon tissue sections.

[0089] Figure 12 shows the change in body weight of mice after treatment with 15% HAMSIAA-0.49.

[0090] Figure 13 shows the change in colon length in mice after treatment with 15% HAMSIAA-0.49.

[0091] Figure 14 shows the change in the DAI index of mice after treatment with 15% HAMSIAA-0.49.

[0092] Figure 15 shows the changes in the colon condition of mice after treatment with 15% HAMSIAA-0.49.

[0093] Figure 16 shows the changes in colon tissue sections of mice after treatment with 15% HAMSIAA-0.49.

[0094] As can be seen from Figures 12 to 16, compared to the control group, the model group (control + DSS) showed a clear decrease in mouse body weight, the HAMS + DSS group showed a reduced decrease, and the 15% HAMSIAA-0.49 + DSS group showed a clear delay in body weight loss, with significant differences observed in both the model group and the HAMS + DSS group (Figure 12). Compared to the control group, the colon length of the model group was significantly shortened, the HAMS group showed no significant difference compared to the model group, and the 15% HAMSIAA group showed a significant decrease in colon length. The HAMSIAA-0.49+DSS group had a significantly longer colon compared to the model group (Figure 13), and the DAI index in the 15% HAMSIAA-0.49+DSS group was significantly reduced compared to the model group (Figure 14). Figure 15 shows colon images of the control group, control+DSS group, HAMS+DSS group, and 15% HAMSIAA-0.49+DSS group, with the 15% HAMSIAA-0.49+DSS group showing the best colonic condition. Compared to the control group, the model group showed more severe colonic tissue damage, with lesions such as glandular loss and extensive necrosis of the mucosal layer. Compared to the model group, the degree of colonic damage was slightly reduced in the HAMS+DSS group, and a small amount of normal intestinal glands were present, but HAMSIAA-0.49 showed the most significant protective effect on mouse colonic tissue (Figure 16). Changes in body weight, colon length, DAI index, and tissue sections all suggested that HAMSIAA could effectively reduce the severity of DSS-induced mouse ulcerative colitis.

[0095] Furthermore, using 1.5% HAMSIAA-0.32 as an example, this application further demonstrates that adding HAMSIAA to feed can effectively reduce the severity of DSS-induced mouse ulcerative colitis by detecting the levels of cytokines such as IL-10, IL-6, IL-1β, and IL-22, as well as the relative expression level of mRNA (AhR / GAPDH) in mouse colon tissue, and that the reduction of DSS-induced mouse ulcerative colitis by HAMSIAA may be closely related to the activation of the AHR / IL-22 pathway. However, Figures 17-19 demonstrate that HAMSIAA effectively reduces the severity of DSS-induced mouse ulcerative colitis from a cytokine level perspective. IL-10, IL-1β is an important anti-inflammatory cytokine secreted by immune and non-immune cells. A correlation was found between the development of colitis and a decrease in IL-10 levels. IL-10 expression was related to disease progression; as the severity of the lesion worsened, IL-10 expression tended to decrease, and then increased during the remission phase, suggesting that IL-10 begins to exert its anti-inflammatory effect, restoring the balance between inflammatory and anti-inflammatory factors. Compared to the model group, the HAMSIAA treatment group showed significantly elevated levels of IL-10 in colonic tissue, demonstrating that HAMSIAA effectively alleviates DSS-induced colitis (Figure 17). IL-6 and IL-1β are important pro-inflammatory cytokines. IL-6 and IL-1β levels were significantly elevated in patients with colitis. Compared to the model group, the HAMSIAA-treated group showed a significant decrease in IL-6 and IL-1β levels in colon tissue, further demonstrating that HAMSIAA effectively alleviates DSS-induced colitis (Figures 18 and 19).

[0096] Figures 20 and 21 show that the improvement in colitis treated with HAMSIAA may be closely related to the activation of the AHR / IL-22 pathway. By detecting the relative expression levels of mRNA (AhR / GAPDH) in colon tissue, it was found that the relative expression of AhR was significantly increased in the HAMSIAA-treated group compared to the model group, indicating that IAA binds to AhR and promotes its expression (Figure 20). By detecting IL-22 levels in colon tissue, it was found that the IL-22 content was significantly increased in the HAMSIAA-treated group compared to the model group (Figure 21). IL-22 has the ability to constitutively activate STAT3 and promotes epithelial cell regeneration and improves the integrity of the mucosal barrier by stimulating the expression of antimicrobial peptides and mucins. IL-22 derived from group 3 innate lymphocytes is useful in the intestinal barrier, and IL-22 expression is regulated by AhR activation. This was strong evidence that IAA binds to AhR. Therefore, improvement in HAMSIAA with treatment of colitis may be closely related to the activation of the AHR / IL-22 pathway.

[0097] Example 7: Reduction of systemic lupus erythematosus (SLE) by HAMSIAA 1. Rat food was prepared using manufactured HAMSIAA at a predetermined additive ratio. SLE model mice and control mice were purchased from Changzhou Kawensi Laboratory Animal Co., Ltd. and were 4-6 weeks old. All mice were randomly divided into three groups. Control group (MRL / MPJ) Systemic lupus erythematosus model group (MRL / lpr) HAMSIAA group (MRL / lpr + HAMSIAA)

[0098] The HAMSIAA group was given a special diet supplemented with HAMSIAA at a predetermined ratio for 14 weeks. Kidney tissue was collected after the experiment was completed.

[0099] 2. In this application, using 1.5% HAMSIAA-0.32 as an example, we have demonstrated that adding HAMSIAA to feed can effectively mitigate SLE based on pathological indicators of the kidneys.

[0100] Figure 22 shows comparative images of HE staining of kidney tissue from each group of mice in this example.

[0101] Figure 23 shows the pathological scores of the kidney tissue of each group of mice in this example.

[0102] Both the HE staining of the mouse kidney tissue and the pathological scores of the kidney tissue described above showed that HAMSIAA significantly reduced glomerulonephritis and interstitial nephritis in mice compared to the SLE mouse model group. These results demonstrate that HAMSIAA can effectively alleviate symptoms in SLE mice.

[0103] Finally, it should be added that the above embodiments are merely for illustrative purposes and do not impose any limitations. Although the present application has been described in detail with reference to the embodiments described above, as those skilled in the art will understand, it is still possible to modify the technical means described in the embodiments described above, or to make equivalent substitutions to some or all of their technical features, and such modifications or substitutions will not cause the essence of the technical means in question to deviate from the scope of the technical means relating to the embodiments of the present application. [Industrial applicability]

[0104] The starch-indolecarboxylic acid derivative provided by this application is an acylated starch formed by the esterification reaction of starch and indolecarboxylic acid. It has high resistance, can withstand degradation in the stomach and small intestine, and can release indolecarboxylic acid after reaching the colon through fermentation by the intestinal flora. It has clear advantages compared to conventional methods of administering indolecarboxylic acid such as intragastric administration and intraperitoneal injection, and can significantly increase the indolecarboxylic acid content in the colon and hepatic portal blood. Furthermore, the starch-indolecarboxylic acid derivative of this application can be widely used in products that regulate the balance of intestinal immunity and products that prevent and treat inflammatory diseases. In addition, in the method for producing the starch-indolecarboxylic acid derivative of this application, starch is dissolved in a solvent to obtain solution A, then indolecarboxylic acid, a condensing agent and a base are added to solution A to carry out an esterification reaction to obtain reaction solution A, then starch in reaction solution A is precipitated, and the starch-indolecarboxylic acid derivative is obtained by suction filtration and drying in that order. The above manufacturing method has the advantage of being simple in its processing steps and suitable for industrial mass production.

Claims

1. A starch-indole carboxylic acid derivative, It is produced by the esterification reaction of starch and indolecarboxylic acid using a condensing agent and a base. The starch-indolecarboxylic acid derivative comprises at least one of indoleacetic acid derivative starch having a degree of substitution of 0.06 to 0.49, and indolepropionic acid derivative starch having a degree of substitution of 0.25 to 0.

39. The indolecarboxylic acid comprises at least one of indoleacetic acid, indolepropionic acid, indoleacrylic acid, or indolelactic acid. The starch comprises at least one of high-amylose corn starch (HAMS), potato starch, sweet potato starch, mixed bean starch, and banana starch. The starch-indole carboxylic acid derivative is a starch in which the high-amylose corn starch has an amylose starch content of more than 50%.

2. The starch-indole carboxylic acid derivative according to claim 1, wherein the condensing agent comprises at least one of EDCI, DCC, and HATU.

3. The starch-indole carboxylic acid derivative according to claim 1, wherein the base comprises at least one of 1-methylimidazole, an amine, sodium bicarbonate, and sodium carbonate.

4. (a) Dissolve starch in a solvent to obtain solution A, then add an indolecarboxylic acid containing at least one of indoleacetic acid, indolepropionic acid, indoleacrylic acid, or indolelactic acid, a condensing agent, and a base to solution A to carry out an esterification reaction to obtain reaction solution A. (b) A method for producing a starch-indolecarboxylic acid derivative according to any one of claims 1 to 3, comprising the step of precipitating starch in reaction solution A, followed by suction filtration, washing, and drying in that order to obtain a starch-indolecarboxylic acid derivative.

5. The method for producing a starch-indolecarboxylic acid derivative according to claim 4, wherein the solvent in step (a) comprises at least one of DMSO, an ionic liquid, and water.

6. The method for producing a starch-indolecarboxylic acid derivative according to claim 4, wherein the temperature of the esterification reaction in step (a) is 20 to 80°C and the duration is 20 to 30 hours.

7. The method for producing a starch-indolecarboxylic acid derivative according to claim 4, wherein the precipitation method in step (b) is to dropwise add reaction solution A to ethanol or water to precipitate the starch-indolecarboxylic acid derivative.

8. Use of the starch-indolecarboxylic acid derivative according to any one of claims 1 to 3 in the manufacture of a product that modulates the balance of intestinal immunity.

9. The use according to claim 8, wherein the adjustment of the balance of intestinal immunity refers to the fact that after the starch-indole carboxylic acid derivative is ingested and reaches the colon, the linked indole carboxylic acid is released by fermentation of the intestinal microbiota, and in synergy with the short-chain fatty acids released by the intestinal microbiota fermentation of starch, synergistically exerting an immunomodulatory effect through multiple immune system signaling pathways.

10. The use according to claim 8, wherein the product that regulates the balance of intestinal immunity includes a drug, a health food, a food for special medical purposes, or a general food.

11. The use according to claim 10, wherein the drug is a drug that prevents and treats inflammatory diseases and / or autoimmune diseases.

12. The use according to claim 11, wherein the inflammatory disease and / or autoimmune disease includes one of inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease, and multiple sclerosis.

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