Starch-indole acid derivatives and their use

Abstract

The application provides a starch-indole acid derivative and a preparation method and application thereof, and relates to the technical field of modified starches. The starch-indole acid derivative refers to esterified starch generated by esterification reaction of starch, a condensing agent and alkali and indole acid. The starch-indole acid derivative has high resistance and can resist degradation of the stomach and small intestine. After reaching the colon site, the starch-indole acid derivative can be fermented by intestinal flora to release indole acid beneficial to intestinal health. Compared with traditional drug delivery modes such as intragastric administration and intraperitoneal injection, the starch-indole acid derivative has obvious advantages and can significantly increase the content of indole acid in the colon and hepatic portal vein blood. In addition, indole acid can activate an aromatic hydrocarbon receptor to play an immunoregulatory role. The immunoregulatory role of indole acid is superimposed on the regulation role of short-chain fatty acids released by starch fermentation by intestinal flora, so that a product playing an immunoregulatory role through multiple immune system signal pathways is provided.

Description

[0001] This application is a divisional application of the invention patent application with the application number 202111213344.7 and the application name "Starch-indole acid derivative and its preparation method and application", the application date of which is October 19, 2021. TECHNICAL FIELD

[0002] The present application relates to the technical field of modified starch, in particular to a starch-indole acid derivative and its preparation method and application. BACKGROUND

[0003] Indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-lactic acid (ILA) and indole-3-acrylic acid (IA) and other indole acid derivatives are intestinal microbial metabolites of tryptophan, which play an important role in regulating intestinal immune balance. Studies have found that IAA, IA, ILA and other substances can act as ligands of aryl hydrocarbon receptor (AhR) to regulate intestinal immune balance, and IAA inhibits the level of pro-inflammatory cytokines in mouse macrophages and hepatocytes in an AhR-dependent manner, thereby relieving liver inflammatory response. IPA regulates mouse intestinal barrier function by acting as a ligand of pregnane X receptor (PXR), especially in the presence of indole, and elevated serum IPA concentration is associated with reduced prevalence of type 2 diabetes, insulin secretion and insulin sensitivity; in addition, IPA can also act as a hydroxyl radical scavenger to exert antioxidant effect.

[0004] Tryptophan is an essential amino acid for humans, which is provided by dietary protein. There are mainly three pathways for the metabolism of tryptophan in the gastrointestinal tract: the kynurenine pathway, the serotonin pathway and direct decomposition by intestinal microorganisms. Intestinal microorganisms are most abundant in the colon, which is the main site for the decomposition of tryptophan to produce IAA / IPA and other indole acids. Studies have found that the content of IAA in the feces of obese and diabetic patients is significantly lower than that in normal people, and epidemiological studies have shown that serum IPA levels are negatively correlated with type 2 diabetes (T2D) and low-grade inflammation, indicating that the content of IAA / IPA and other indole acids is closely related to human diseases. Therefore, targeting the colon to deliver IAA / IPA and other indole acids is expected to take advantage of the good immune regulation effect of IAA / IPA and other indole acids, thereby preventing or treating inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease, multiple sclerosis and other inflammatory and autoimmune diseases.

[0005] However, if these small indole acid molecules are directly ingested orally, they will be absorbed in the stomach or small intestine of the digestive tract and cannot effectively reach the colon site. How to target the delivery of these indole acid small molecules with immune regulation effect to the colon site and exert their effect is a problem to be solved.

[0006] In view of the above, the present application is proposed. SUMMARY

[0007] A first object of the present application is to provide a starch-indole acid derivative which can resist degradation in the stomach and small intestine, release indole acid through intestinal flora fermentation after reaching the colon site, and has obvious advantages compared with traditional indole acid administration methods such as intragastric administration and intraperitoneal injection, and can significantly increase the content of indole acid in the colon and hepatic portal vein blood.

[0008] A second object of the present application is to provide a preparation method of the starch-indole acid derivative.

[0009] A third object of the present application is to provide an application of the starch-indole acid derivative, which can be widely applied in the preparation process of intestinal immune balance regulating products.

[0010] In order to achieve the above objects of the present application, the following technical solutions are adopted:

[0011] The starch-indole acid derivative provided by the present application is mainly prepared by esterification of starch, a condensing agent and an alkali, and indole acid.

[0012] Preferably, the indole acid substitution degree of the starch-indole acid derivative after condensation is 0.01-1.0.

[0013] Further, the indole acid includes at least one of indole acetic acid, indole propionic acid, indole propylene acid and indole lactic acid.

[0014] Further, the starch includes at least one of high amylose corn starch (HAMS), potato starch, sweet potato starch, mixed bean starch and banana starch.

[0015] Further, the condensing agent includes at least one of EDCI, DCC and HATU.

[0016] Preferably, the alkali includes at least one of 1-methylimidazole, amine, sodium bicarbonate and sodium carbonate.

[0017] The preparation method of the above starch-indole acid derivative provided by the present application includes the following steps:

[0018] (a) dissolving starch in a solvent to obtain solution A; then adding indole acid, a condensing agent and an alkali into solution A to perform esterification reaction, and obtaining reaction liquid A;

[0019] (b) precipitating starch in reaction liquid A, and then sequentially performing suction filtration and drying to obtain the starch-indole acid derivative.

[0020] Further, the solvent in the step (a) comprises at least one of DMSO, ionic liquid and water.

[0021] Further, the temperature of the esterification reaction in the step (a) is 20-80 DEG C, and the time is 20-30 h.

[0022] Further, the method for precipitating in the step (b) is that the reaction liquid A is added dropwise into ethanol or water, and the starch-indole acid derivative is precipitated.

[0023] The starch-indole acid derivative provided by the application is used for preparing a product for regulating intestinal immune balance.

[0024] The regulation of intestinal immune balance refers to that after the starch-indole acid derivative is taken and reaches the colon part, the connected indole acid is released through fermentation of intestinal flora, cooperates with short-chain fatty acid released by fermentation of starch through intestinal flora, and cooperatively plays an immune regulation role through multiple immune system signal paths.

[0025] Further, the product for regulating intestinal immune balance comprises medicine, health food, special medical purpose formula food or ordinary food.

[0026] Further, the medicine is a medicine for preventing and treating inflammatory diseases and / or autoimmune diseases.

[0027] Further, the inflammatory diseases and / or autoimmune diseases comprise one of inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease and multiple sclerosis.

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] The starch-indole acid derivative provided by the application is mainly prepared by esterification of starch and indole acid under the action of a condensing agent and alkali; the starch-indole acid derivative is acylated starch formed by esterification of starch and indole acid, has high resistance, can resist degradation of the stomach and small intestine, can release indole acid through fermentation of intestinal flora after reaching the colon part, has obvious advantages compared with traditional indole acid administration modes such as gavage and intraperitoneal injection, and can significantly improve the content of indole acid in the colon and hepatic portal vein blood.

[0030] The indole acid released by the starch-indole acid derivative provided by the application in the colon part plays an immune regulation role by activating AhR. Meanwhile, the short-chain fatty acid produced by fermentation of starch by intestinal flora can also play an immune regulation role by activating G protein-coupled receptors and inhibiting histone deacetylase. The effects of indole acid and short-chain fatty acid are positively superimposed, and a product for cooperatively playing an immune regulation role through multiple immune system signal paths can be provided.

[0031] The starch-indole acid derivative preparation method provided by the present application comprises the following steps: dissolving starch in a solvent to obtain solution A; then adding indole acid, a condensing agent and an alkali into solution A to perform an esterification reaction, so as to obtain reaction liquid A; and then precipitating starch in reaction liquid A, and sequentially performing suction filtration and drying, so as to obtain the starch-indole acid derivative. The preparation method has the advantages of simple processing technology and suitability for industrial large-scale production.

[0032] The starch-indole acid derivative provided by the present application can be widely applied in products for regulating intestinal immune balance, and products for preventing and treating inflammatory diseases. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0034] Figure 1 The nuclear magnetic hydrogen spectrum of the starch-indole acid derivative provided for example 1 of the present application;

[0035] Figure 2 The infrared spectrum of starch (HAMS) and indole acetic acid derivative high straight chain corn starch (HAMSIAA) provided for example 1 of the present application;

[0036] Figure 3 The XRD spectrum of starch (HAMS) and indole acetic acid derivative high straight chain corn starch (HAMSIAA) provided for example 1 of the present application;

[0037] Figure 4 The SEM electron microscope graph of starch (HAMS) and indole acetic acid derivative high straight chain corn starch (HAMSIAA) provided for example 1 of the present application;

[0038] Figure 5 The concentration graph of indole acid in the feces of experimental mice provided for example 2 of the present application;

[0039] Figure 6 The concentration graph of indole acid in the hepatic portal vein blood of experimental mice provided for example 2 of the present application;

[0040] Figure 7 The concentration graph of indole acid in the feces of experimental mice provided for example 3 of the present application;

[0041] Figure 8The concentration of IAA in the colon contents of different administration modes provided for Example 4 of the present application;

[0042] Figure 9 The concentration of IAA in the serum of the hepatic portal vein of different administration modes provided for Example 4 of the present application;

[0043] Figure 10 The concentration of IPA in the feces of the experimental mice provided for Example 5 of the present application;

[0044] Figure 11 The IAA / IPA concentration ratio chart in the serum of the peripheral blood of the normal group and the DSS-induced ulcerative colitis group provided for Example 6 of the present application;

[0045] Figure 12 The body weight change chart of the mice after induction of 15% HAMS IAA-0.49 provided for Example 6 of the present application;

[0046] Figure 13 The colon length change chart of the mice after induction of 15% HAMS IAA-0.49 provided for Example 6 of the present application;

[0047] Figure 14 The DAI index change chart of the mice after induction of 15% HAMS IAA-0.49 provided for Example 6 of the present application;

[0048] Figure 15 The colon state change chart of the mice after induction of 15% HAMS IAA-0.49 provided for Example 6 of the present application;

[0049] Figure 16 The colon tissue section change chart of the mice after induction of 15% HAMS IAA-0.49 provided for Example 6 of the present application. DETAILED DESCRIPTION

[0050] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0051] According to an aspect of the present application, a starch-indole acid derivative is prepared mainly from amylose by esterification reaction with indole acid under the action of a condensing agent and an alkali;

[0052] Preferably, the indole acid substitution degree of the starch-indole acid derivative after condensation is 0.01-1.0.

[0053] The starch-indole acid derivative is mainly prepared by esterification of starch and indole acid under the action of a condensing agent and a base; the starch-indole acid derivative is acylated starch formed by esterification of starch and indole acid, has high resistance, can resist degradation of the stomach and small intestine, and can release indole acid through intestinal flora fermentation after reaching the colon site, thus having obvious advantages over traditional indole acid administration modes such as intragastric administration and intraperitoneal injection, and can significantly improve the content of indole acid in the colon and hepatic portal vein blood.

[0054] It should be noted that targeted release of indole acid to the colon site can effectively prevent and treat inflammatory diseases and autoimmune diseases; the inflammatory diseases and autoimmune diseases include, but are not limited to, inflammatory bowel disease, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune liver disease, multiple sclerosis, etc.

[0055] Preferably, the starch-indole acid derivative includes indole acid derivatized starch, and is a mixture of one or more of indole acetylated starch, indole propionylated starch, indole lactylated starch and indole acrylated starch.

[0056] In a preferred embodiment of the present application, the indole acid includes at least one of indole acetic acid, indole propionic acid, indole propenoic acid and indole lactic acid.

[0057] In a preferred embodiment of the present application, the starch includes at least one of high amylose corn starch (HAMS), potato starch, sweet potato starch, mixed bean starch and banana starch.

[0058] It should be noted that the high amylose starch refers to starch with a content of amylose exceeding 50%.

[0059] In a preferred embodiment of the present application, the condensing agent includes at least one of EDCI, DCC and HATU.

[0060] In a preferred embodiment of the present application, the base includes at least one of 1-methylimidazole, amine, sodium bicarbonate and sodium carbonate.

[0061] According to an aspect of the present application, a preparation method of the above starch-indole acid derivative includes the following steps:

[0062] (a) dissolving starch in a solvent to obtain solution A; then adding indole acid, a condensing agent and a base into solution A to perform esterification, and obtaining reaction liquid A;

[0063] (b) precipitating starch in reaction liquid A, and then sequentially performing suction filtration and drying to obtain the starch-indole acid derivative.

[0064] The starch-indole acid derivative preparation method provided by the present application comprises the following steps: dissolving starch in a solvent to obtain solution A; then adding indole acid, a condensing agent and a base into solution A to perform esterification reaction, and obtaining reaction liquid A; and then precipitating starch in reaction liquid A, and sequentially performing suction filtration and drying to obtain the starch-indole acid derivative. The preparation method has the advantages of simple processing technology and suitability for industrial large-scale production.

[0065] In a preferred embodiment of the present application, the solvent in step (a) comprises at least one of DMSO, an ionic liquid and water.

[0066] In a preferred embodiment of the present application, the esterification reaction in step (a) is performed at a temperature of 20-80°C for 20-30 hours.

[0067] In a preferred embodiment of the present application, the precipitation method in step (b) comprises dropping reaction liquid A into ethanol or water to precipitate the starch-indole acid derivative.

[0068] According to an aspect of the present application, the starch-indole acid derivative is used for preparing a product for regulating intestinal immune balance.

[0069] The starch-indole acid derivative provided by the present application can be widely applied to the preparation process of the product for regulating intestinal immune balance.

[0070] In a preferred embodiment of the present application, the regulation of intestinal immune balance refers to that the starch-indole acid derivative is used as a targeted carrier to deliver indole acid to the colon.

[0071] Preferably, the product for regulating intestinal immune balance comprises a medicine, a health food, a special medical purpose formula food or a general food.

[0072] The technical solutions of the present application will be further described below with reference to the embodiments.

[0073] Embodiment 1

[0074] (I) Preparation of starch-indole acid derivative

[0075] (1) Preparation of high straight-chain corn starch derivatized with indole acetic acid (HAMSIAA)

[0076] (2) Preparation of high straight-chain corn starch derivatized with indole propionic acid (HAMSIPA)

[0077] HAMS was added to DMSO, stirred until the solution was clear, IPA, EDCI, 1-methylimidazole were added in turn, after the addition was completed, the stirring was continued for 24 hours. After the reaction was completed, the reaction solution was added dropwise into EtOH or H2O, the solid was precipitated, suction filtered, the filter cake was washed with EtOH or H2O, and dried.

[0078] By adjusting the feeding ratio, HAMSIAA and HAMSIPA with different degrees of substitution (0.01-1.0) were obtained. Taking HAMSIAA as an example (Table 1):

[0079] Table 1 Preparation of HAMSIAA with different degrees of substitution

[0080]

[0081] (3) Determination of the degree of substitution of indole acid derivatized high straight chain corn starch:

[0082] The determination of the degree of substitution of indole acid derivatized high straight chain corn starch by titration includes two steps of alkali hydrolysis of ester bond and neutralization of excess alkali, and the specific operation is as follows:

[0083] 0.2 g of starch was weighed, 10 mL of acetone and 1 mL of water were added, sealed, and magnetically stirred until mixed evenly; 3 mL of NaOH aqueous solution (1 mol / L) was added, and magnetically stirred for 30 min; 10 mL of hot water at 60°C was added to rinse the bottle wall, and the stirring was continued for 2 min; cooled, 3 drops of phenolphthalein reagent were added, and titrated with HCl (0.5 mol / L) until the solution became colorless, and the volume of HCl was recorded. The determination was carried out in triplicate.

[0084] The mass fraction ω of indole acetyl group in indole acid derivatized high straight chain corn starch HAMSIAA is:

[0085]

[0086] In the formula, V2 and V1 are the volumes (mL) of HCl used in the titration of HAMS and HAMSIAA respectively, c is the concentration (mol / L) of HCl, m is the mass (g) of the HAMSIAA sample, and 158 is the molecular mass of indole acetyl group.

[0087] The degree of substitution DS calculated from the content of indole acetyl group is as follows:

[0088]

[0089]

[0090] 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.

[0091] Similarly, the degree of substitution of HAMSIPA is shown below:

[0092]

[0093]

[0094] In the formula, ω is the mass fraction of indolepropionyl group in modified starch HAMSIPA, V2 and V1 are the volumes (mL) of HCl used in the titration of HAMS and HAMSIPA, respectively, c is the concentration of HCl (mol / L), m is the mass (g) of HAMSIPA sample, 172 is the relative molecular mass of indolepropionyl group, and 162 is the relative molecular mass of each glucose unit of starch.

[0095] (4) Structural characterization of indole acid-derived starch:

[0096] By nuclear magnetic resonance hydrogen spectrum ( 1 The specific structural information of the modified starch was characterized by ¹H NMR and FTIR spectra. The crystallinity of the modified starch was determined by X-ray diffraction (XRD), and the ultrastructure of the starch granules was analyzed by scanning electron microscopy (SEM).

[0097] Figure 1 The above is the 1H NMR spectrum of the starch-indole acid derivative provided in this embodiment; by Figure 1 It can be seen that, compared with HAMS, HAMSIAA and HAMSIPA have a characteristic NH peak at a chemical shift of 10.83 ppm, and HAMSIPA has two new peaks at 2.96 ppm and 2.69 ppm, which are characteristic peaks of two methylene groups in IPA.

[0098] Figure 2 The infrared spectrum of starch (HAMS) and indoleacetic acid-derived high amylose corn starch (HAMSIAA) provided in this embodiment; by Figure 2 It can be known that 400cm -1 The peak at 2930 cm⁻¹ is a stretching vibration peak of OH or NH. The OH peak is relatively broad, while the NH peak is relatively sharp. Because IAA contains NH, the peaks become more and more pronounced with increasing degree of substitution. -1 The peak at 1728 cm⁻¹ represents the stretching vibration peak of CH. Compared to HAMS, HAMSIAA has a peak at 1728 cm⁻¹. -1 A new peak appears at 1728 cm⁻¹, which is the stretching vibration peak of the carbonyl group. -1 The carbonyl peak at 745 cm⁻¹ becomes stronger with increasing degree of substitution. This can be seen from the spectrum.-1 The peak at 1728cm-1, which is the out-of-plane bending vibration peak of the aromatic ring CH, also becomes stronger with the increase of the degree of substitution of HAMSIAA. The appearance of the carbonyl characteristic peak at 1728cm-1 and the aromatic ring CH characteristic peak at 745cm-1 indicates that the esterification reaction between HAMS and IAA is successful. -1 -1 The appearance of the carbonyl characteristic peak at 1728cm-1 and the aromatic ring CH characteristic peak at 745cm-1 indicates that the esterification reaction between HAMS and IAA is successful.

[0099] Note: Figure 2 DS in the formula is the degree of substitution.

[0100] It should be noted that according to literature reports, starch granules are a polymorphic system, and the crystal structure of starch granules varies with different plant varieties, mainly producing three types of X-ray diffraction patterns (type A, type B, and type C). In addition, type V structure can be obtained by special methods such as starch acylation, and some genetically bred starches show A+V, B+V, and C+V types. Starches of different crystal forms have obvious characteristic peaks, type A has strong peaks at 15°, 17°, 18°, and 23°; type B has strong peaks at 5.6°, 17°, 22°, and 24°; type C shows a combination of type A and type B, with a peak at 5.6° compared to type A, and a strong peak at 23° compared to type B; the characteristic peaks of type V are 12.5° and 19.5°.

[0101] Figure 3 The XRD pattern of starch (HAMS) and indole acetic acid derivatized high amylose corn starch (HAMSIAA) provided for this example; from Figure 3 It can be seen that HAMS has peaks at 5.6°, 17°, 19.5°, and 22°, which is a B+V type crystal structure; and HAMSIAA has characteristic peaks at 12.5° and 19.5°, which is a V type, and the peaks become weaker and weaker with the increase of the degree of substitution. These results show that the acylation of HAMS destroys its crystal structure.

[0102] Figure 4 The SEM electron micrograph of starch (HAMS) and indole acetic acid derivatized high amylose corn starch (HAMSIAA) provided for this example; from Figure 4 It can be seen that HAMS starch granules are mostly round or oval, with smooth surfaces and no cracks, while the surface of HAMSIAA starch granules after IAA acylation becomes rough and irregular, because during the reaction process, DMSO is used for dissolution, and after the reaction is completed, the product is precipitated by adding ethanol or water, which destroys the granular morphology of the starch.

[0103] Example 2 Evaluation of the colon-targeted delivery effect of indole acetic acid derivatized starch (HAMSIAA):

[0104] ​HAMS and HAMSIAA with different degrees of substitution were added to the mouse diet at a ratio of 15%. Eight-week-old mice were randomly divided into 6 groups of 5 mice each according to their body weight: HAMS group and HAMSIAA group (6 groups, DS values ​​of 0.065, 0.12, 0.19, 0.29, 0.37, and 0.49, respectively). Mouse feces and serum were collected.

[0105] (1) Methods for determining the IAA / IPA content in mouse feces, portal vein blood, peripheral blood and colon tissue;

[0106] The contents of the metabolites IAA / IPA in each sample were determined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). For the determination of IAA / IPA content in mouse feces: Methanol was added to the collected feces, followed by vortexing, ultrasonic extraction, and centrifugation. The supernatant was collected as the extract. The extract was then processed, and IAA / IPA internal standards (IAA-d5 / IPA-d2) were added for correction. The extract was then purified using an HLB solid-phase extraction column (pretreated with 5 mL methanol and 5 mL water, then rinsed and discarded with a 5 mL methanol / water mixture and 5 mL water, and finally eluted with 5 mL methanol). The eluent was then analyzed by HPLC-MS / MS to detect IAA / IPA. Determination of IAA / IPA content in portal vein and peripheral blood: Whole blood samples were allowed to stand and centrifuged at low temperature. The supernatant was collected to obtain serum. The same method as above was used, with an internal standard added to the serum, followed by purification via solid-phase extraction. The IAA / IPA metabolites in the eluent were detected using HPLC-MS / MS. Determination of IAA / IPA content in colon tissue: Methanol was added to the collected colon tissue, which was then ultrasonically broken down and further ground until homogeneous. The tissue was then vortexed, ultrasonically extracted, and centrifuged. The supernatant was collected to obtain the extract. The same method as above was used, with an internal standard added to the serum, followed by purification via solid-phase extraction. The IAA / IPA concentration in the extract was detected using HPLC-MS / MS.

[0107] Figure 5 This is a graph showing the concentration of indoleic acid in the feces of the mice used in this experiment. Figure 5 It was found that when the modified starch content in the feed was 15%, the HAMSIAA group significantly increased the IAA concentration in feces compared to the HAMS group. Specifically, the highest IAA concentration in feces was observed when the degree of substitution of HAMSIAA was 0.37, approximately 5470 μmol / kg, which was about 1370 times higher than that of the HAMS group (approximately 4 μmol / kg). This indicates that the carrying capacity of IAA is related to the degree of substitution; when the degree of substitution is low, the IAA concentration in feces increases with increasing substitution, but once the degree of substitution reaches a certain value, further increases lead to a decrease in IAA concentration.

[0108] Figure 6 Figure 2 is a graph showing the concentration of indole acid in the liver portal vein blood of mice in the experiment of this embodiment. It can be seen that, when the amount of modified starch added to the feed is 15%, the HAMSIAA group can significantly increase the concentration of IAA in the liver portal vein blood of mice compared with the HAMS group. The change rule of the concentration of IAA in the liver portal vein blood of different substitution degree HAMSIAA groups is slightly different from that in the feces. Among them, the HAMSIAA group with a substitution degree of 0.19 has the highest concentration of IAA in the liver portal vein blood, about 2700 nmol / mL, which is about 2700 times higher than that of the HAMS group (about 1 nmol / mL); while the HAMSIAA group with a substitution degree of 0.37 has the highest concentration of IAA in the feces, which may be because the resistance of the HAMSIAA with a substitution degree of 0.37 is stronger, less absorption in the stomach or small intestine, and more decomposition and absorption in the colon. Figure 6 By detecting the concentration of IAA in the feces and liver portal vein blood of mice, it is found that, when the amount of modified starch added to the feed is 15%, the HAMSIAA group can significantly increase the concentration of IAA compared with the HAMS group, which indicates that the HAMSIAA can well target and slowly release IAA to the colon, and the HAMSIAA with a substitution degree of 0.37 has the best targeting and transporting effect.

[0109] Example 3 Evaluation of the colon-targeting and transporting effect of indole acetic acid derivatized starch (HAMSIAA)

[0110] HAMS and HAMSIAA with different substitution degrees were respectively added to the mouse feed at a proportion of 1.5%, and 8-week-old mice were randomly divided into 6 groups according to the body weight, 5 mice in each group: HAMS group, HAMSIAA group (6 groups, DS is 0.065, 0.12, 0.19, 0.29, 0.37, 0.49), and the feces and serum of mice were collected.

[0111]

[0112] Figure 3 is a graph showing the concentration of indole acid in the feces of mice in the experiment of this embodiment. It can be seen that, when the amount of modified starch added to the feed is reduced to 1.5%, the HAMSIAA group can still significantly increase the concentration of IAA in the feces compared with the HAMS group, and the HAMSIAA group with a substitution degree of 0.37 has the highest concentration of IAA in the feces, about 860 μmol / kg, which is about 200 times higher than that of the HAMS group (about 4 μmol / kg). Figure 7 Figure 7 Example 4 Evaluation of the targeting and transporting effect of different administration methods

[0113] Example 4 Evaluation of the targeting and transporting effect of different administration methods

[0114] ​The experiment was divided into 5 groups:

[0115] 1. Blank control group;

[0116] 2. HAMS IAA-0.32 (1.5%) group (3 g of mouse feed was consumed by each mouse per day, and a total of 0.068 mmol of IAA was ingested);

[0117] 3. IAA Na drinking water group (the concentration of IAA Na was 2.68 mg / mL, 5 mL of water was drunk by each mouse per day, and a total of 0.068 mmol of IAA Na was ingested);

[0118] 4. Intragastric administration group (the concentration of IAA Na was 67 mg / mL, 0.2 mL was administered per day, and a total of 0.068 mmol of IAA Na was ingested);

[0119] 5. Intraperitoneal injection group (the concentration of IAA Na was 67 mg / mL, 0.2 mL was injected per day, and a total of 0.068 mmol of IAA Na was ingested).

[0120] By comparing the IAA concentrations in the colon contents and the hepatic portal vein of mice in the four administration methods of intragastric administration, intraperitoneal injection, direct drinking of IAA salt water, and HAMS IAA, the colon targeting delivery effect of IAA in different administration methods was evaluated, and the results are shown in Figure 8 .

[0121] Figure 8 The IAA concentrations in the colon contents of different administration methods are shown in .

[0122] It can be seen from Figure 8 that compared with the three administration methods of intragastric administration, intraperitoneal injection, and direct drinking of IAA salt water, HAMS IAA can more effectively deliver IAA to the colon site. When the IAA intake is equivalent, the HAMS IAA group significantly increases the IAA concentration in feces by about 200 times compared with the control group; the intragastric administration group and the intraperitoneal injection group have similar IAA concentrations in feces, which are about 7 times higher than the control group; the IAA Na drinking water group has an IAA concentration in feces that is about 30 times higher than the control group. The experimental results show that whether it is intragastric administration or direct drinking of IAA salt water, most of it will be absorbed in the stomach or small intestine, and only a small part can reach the colon site, while HAMS IAA can resist the digestion of the stomach and small intestine by using resistant starch, reach the colon site, and slowly release IAA in the colon site through intestinal microbial fermentation. By detecting the IPA concentrations in the colon contents of mice in different administration methods, it was found that the IPA concentrations in the intragastric administration group, the intraperitoneal injection group, and the IAA Na drinking water group did not change significantly compared with the control group, while the IPA concentration in the HAMS IAA group was significantly higher than that in the control group.

[0123] Figure 9The concentration of IAA in portal vein serum under different administration routes; by Figure 9 It was found that when the IAA intake was comparable, the concentration of IAA in the portal vein blood of mice was significantly increased in all different administration methods compared to the control group. Specifically, there were no significant differences in serum IAA concentration in the gavage group, intraperitoneal injection group, and IAANa drinking water group, while the serum IAA concentration in the 1.5% HAMSIAA-0.32 group was approximately 10-fold higher than that in the other administration methods. This indicates that HAMSIAA has significant advantages in targeted delivery and absorption compared to other administration methods.

[0124] Example 5: Evaluation of the colon-targeted delivery of IPA using indolepropionic acid-derived starch (HAMSIPA):

[0125] Indole-derived starches with different degrees of substitution were added to mouse diets at ratios of 15% and 1.5%, respectively. Mice were fed these specially prepared diets for one week, and fecal samples were collected to detect IPA content. Results are as follows... Figure 10 As shown.

[0126] Figure 10 This is a graph showing the concentration of IPA in the feces of experimental mice provided in this embodiment.

[0127] Depend on Figure 10 It was found that compared with the HAMS group, the fecal IPA concentration in the HAMSIPA group was significantly increased, exhibiting both degree of substitution and dose-dependent effects; the higher the amount added, the higher the fecal IPA concentration. Specifically, the HAMSIPA group with a degree of substitution of 0.25 had the highest fecal IPA concentration. This indicates that HAMSIPA also possesses good colon-targeted IPA delivery capabilities.

[0128] Example 6: HAMSIAA alleviates DSS-induced acute ulcerative colitis:

[0129] The prepared HAMSIAA was used to prepare mouse diets at a specific addition ratio. Mice were divided into control group, HAMS group, control group + DSS group, HAMS + DSS group, and groups with different degrees of substitution of HAMSIAA + DSS. After the start of the experiment, the changes in fecal morphology, mental state, and weight of the mice were observed and recorded daily. The drinking water and DSS solution were changed every 3 days, and on the 7th day after the start of the experiment, the DSS solution was replaced with drinking water.

[0130] After the experiment, the disease active index (DAI) of the mice was assessed, the length of the mouse colon was measured, and the colonic condition was evaluated by sectioning. Finally, these indicators were used to evaluate the preventive and therapeutic effects of HAMSIAA on DSS-induced acute ulcerative colitis in mice.

[0131] Figure 11 Figure 1 is a graph showing the concentration of IAA / IPA in the peripheral blood serum of the normal group and the DSS-induced ulcerative colitis group;

[0132] As can be seen from Figure 11 Compared with the control group, the concentration of IAA / IPA in the serum of the control+DSS group was significantly reduced, indicating that the tryptophan intestinal microbial metabolite IAA / IPA is related to ulcerative colitis, and therefore we speculate that the severity of DSS-induced ulcerative colitis in mice can be alleviated by supplementing IAA / IPA.

[0133] Further, the present application takes 15% HAMS IAA-0.49 as an example, through the indicators of changes in mouse body weight, colon length, DAI index, colon state and colon tissue sections, it is proved that adding HAMS IAA in feed can effectively alleviate the severity of DSS-induced ulcerative colitis in mice, wherein:

[0134] Figure 12 Figure 2 is a graph showing the changes in mouse body weight after induction of 15% HAMS IAA-0.49;

[0135] Figure 13 Figure 3 is a graph showing the changes in colon length of mice after induction of 15% HAMS IAA-0.49;

[0136] Figure 14 Figure 4 is a graph showing the changes in DAI index of mice after induction of 15% HAMS IAA-0.49;

[0137] Figure 15 Figure 5 is a graph showing the changes in colon state of mice after induction of 15% HAMS IAA-0.49;

[0138] Figure 16 Figure 6 is a graph showing the changes in colon tissue sections of mice after induction of 15% HAMS IAA-0.49;

[0139] As can be seen from Figures 12 to 16 Compared with the control group, the body weight of the model group (control+DSS) mice decreased significantly, the HAMS+DSS group was alleviated, and the 15% HAMS IAA-0.49+DSS group significantly slowed down the decrease in body weight, which had significant differences with the model group and the HAMS+DSS group Figure 12 Compared with the control group, the colon length of the model group was significantly shortened, the HAMS group had no significant difference with the model group, while the 15% HAMS IAA-0.49+DSS group had a longer colon length compared with the model group, and had a significant difference Figure 13 Compared with the model group, the DAI index of the 15% HAMS IAA-0.49+DSS group also decreased significantly, and had a significant difference Figure 14 Figure 15 ​For the control group, control+DSS group, HAMS+DSS group, 15% HAMSIAA-0.49+DSS group colon pictures, 15% HAMSIAA-0.49+DSS group of colon state is best. Compared with the control group, the colon tissue damage of the model group is serious, and the gland disappears and large range of mucosal layer necrosis and other lesions appear, compared with the model group, the colon damage degree of HAMS+DSS group is slightly reduced, there are a small amount of normal structure of intestinal gland, and the protective effect of HAMSIAA-0.49 on mouse colon tissue is most obvious. Figure 16 Body weight change, colon length, DAI index and tissue section all show that HAMSIAA can effectively alleviate the severity of DSS-induced ulcerative colitis in mice.

[0140] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of preparing a starch-indole acid derivative, wherein, The starch-indole acid derivative includes indole acetic acid derivatized starch with a degree of substitution of 0.06-0.49, and indole propionic acid derivatized starch with a degree of substitution of 0.25-0.39, the starch including at least one of high amylose corn starch, potato starch, sweet potato starch, bean starch and banana starch, the high amylose corn starch being starch with a content of amylose over 50%, and the method includes the following steps: (a) dissolving starch in a solvent to obtain solution A; then adding indole acid, a condensing agent and a base into solution A to perform esterification, to obtain reaction liquid A; the esterification is performed at a temperature of 20-80℃ for 20-30 hours; (b) precipitating starch from reaction liquid A, then sequentially performing suction filtration, washing and drying to obtain starch-indole acid derivative; the precipitation method is to drop reaction liquid A into ethanol or water to precipitate the starch-indole acid derivative, perform suction filtration, wash the filter cake with ethanol or water, and dry.

2. The method of claim 1, wherein, The condensing agent includes at least one of EDCI, DCC and HATU; And / or, the base includes at least one of 1-methyl imidazole, amine, sodium bicarbonate and sodium carbonate.

3. The method of claim 1, wherein, The solvent in step (a) includes at least one of DMSO, ionic liquid and water.

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