An oral preparation of long-branched starch with function of relieving colitis

Abstract

This invention discloses an oral preparation of long-branched starch with colitis-relieving function, belonging to the field of functional food technology. The alleviating effect of long-branched starch on colitis in mice was further investigated by combining clinical characteristics and histopathological changes. Experimental results show that long-branched starch can improve DSS-induced colonic inflammation and reduce colonic tissue damage; therefore, long-branched starch can be used as a good functional food for relieving colitis.

Description

Technical Field

[0001] This invention relates to an oral preparation of long branched-chain starch with the function of relieving colitis, belonging to the field of functional food technology. Background Technology

[0002] Ulcerative colitis is a chronic, relapsing inflammatory disease primarily affecting the colonic mucosa. Its exact etiology and pathogenesis are not fully understood, but it is generally believed to be related to the interaction of genetic susceptibility, immune system dysregulation, gut microbiota imbalance, and environmental factors. Clinical manifestations are mainly persistent or recurrent diarrhea, mucus-containing bloody stools, and abdominal pain, often accompanied by tenesmus. Some patients may experience systemic symptoms such as weight loss and fatigue. Epidemiological data shows that this disease is common in Europe and the United States, resulting in a heavy disease burden. In recent years, with changes in lifestyle and the widespread availability of diagnostic technology in my country, its prevalence has also shown a significant upward trend, becoming a major public health issue. The disease has a prolonged course and is prone to relapse; long-term active inflammation may also increase the risk of colorectal cancer, seriously affecting patients' quality of life and long-term prognosis.

[0003] In clinical treatment, current first-line regimens mainly include aminosalicylic acid preparations, glucocorticoids, and immunosuppressants, aiming to suppress abnormal immune responses and control inflammatory activity. Although these drugs can induce disease remission in the short term, long-term use often faces problems such as diminished efficacy, high relapse rates, and various adverse reactions (e.g., liver and kidney damage, metabolic disorders, increased risk of infection). Therefore, developing naturally derived active ingredients or functional food ingredients with significant anti-inflammatory activity, good safety profile, and low toxicity is of significant research value and application potential for revolutionizing intervention strategies for ulcerative colitis and meeting the needs of long-term clinical management. Summary of the Invention

[0004] The technical problem that this invention aims to solve is to analyze the main anti-inflammatory components in long amylopectin and provide an anti-inflammatory product that can effectively relieve or help improve colitis with few side effects.

[0005] To address the aforementioned problems, this invention provides a method for preparing long-chain amylopectin, and analyzes the main components that exert anti-inflammatory activity through clinical evaluation. This invention treats DSS-induced colitis in mice using enzymatically modified ordinary corn starch. Clinical evaluation shows that long-chain amylopectin can improve disease activity score (DAI), reduce colonic inflammatory contracture, and improve colonic tissue pathological structure and score, thus achieving an anti-inflammatory effect. The first objective of this invention is to provide a long amylopectin with the following chain length distribution percentages: DP1-5, 0.35%; DP6-12, 3.45%; DP13-24, 41.86%; DP25-36, 48.44%; DP≥37, 5.92%.

[0006] A second objective of this invention is to provide a method for the efficient preparation of long-branched starch using Np-AS enzyme, comprising the following steps: (1) Pretreatment: Mix starch with water to prepare starch emulsion. Heat the starch emulsion to gelatinize it and then cool it to room temperature. Add four times the volume of sucrose and wait for the sucrose to dissolve completely before placing it in a constant temperature water bath at 40°C for 10 min.

[0007] (2) Enzymatic hydrolysis: Add starch sucrase Np-AS from Neisseria polysaccharea and continue enzymatic hydrolysis for 10-20 h. After the hydrolysis is completed, add three times the volume of anhydrous ethanol to the system to terminate the reaction. The product is collected after being repeatedly washed with deionized water and centrifuged three times.

[0008] (3) Product collection for later use: The product obtained after enzymatic hydrolysis is placed in a -80℃ freezer and then freeze-dried for 48 h.

[0009] In one embodiment of the present invention, during pretreatment, the starch in the starch aqueous solution is selected from ordinary corn starch.

[0010] In one embodiment of the present invention, the starch sucrase is Np-AS derived from Neisseria polysacchare, and the amino acid sequence of the enzyme Np-AS is shown in SEQ ID NO.2; the nucleotide sequence encoding the Np-AS enzyme is shown in SEQ ID NO.1.

[0011] In one embodiment of the present invention, the mass concentration of the starch emulsion in step (1) is at least 10% (w / w). Furthermore, sucrose is added after the starch emulsion has cooled to prevent sucrose decomposition.

[0012] In one embodiment of the present invention, after Np-AS enzymatic hydrolysis, three times the volume of anhydrous ethanol is added to inactivate the enzyme and terminate the reaction. The product is then washed three times with deionized water, primarily to remove small sugar molecules such as fructose, thus avoiding interference with the determination of the resistant starch content in the product.

[0013] In one embodiment of the present invention, step (3) is to boil and inactivate the enzyme for 15 minutes and then freeze.

[0014] In one embodiment of the present invention, in step (2), the amount of starch sucrase added is 50~250 U / g dry starch.

[0015] In one embodiment of the present invention, during the enzymatic hydrolysis process, a stirrer and a four-necked flask are used for stirring at a speed of 300 r / min.

[0016] A third objective of this invention is to provide the use of long-branched starch in the preparation of medicines for relieving or assisting in the improvement of colitis.

[0017] In one embodiment of the present invention, the dosage of long-chain starch is 100-200 mg / kg.

[0018] In one embodiment of the present invention, the dosage form of the drug includes tablets, capsules, granules, injections, liposome nanoparticles, sustained-release agents, dispersible tablets, or enteric-coated granules.

[0019] In one embodiment of the present invention, the medicine further comprises a pharmaceutical carrier and / or pharmaceutical excipients.

[0020] A fourth objective of the present invention is to provide an oral formulation of the long amylopectin, characterized in that the oral formulation comprises 75-80% w / w long amylopectin, 17-20% w / w microcrystalline cellulose, and 2-4% w / w magnesium stearate, which is prepared by tableting, encapsulating, granulating, and drying.

[0021] Beneficial effects: This invention uses a mouse model of colitis induced by dextran sulfate sodium (DSS) to investigate the alleviating effect of principal components on intestinal inflammation in model mice, based on disease activity score (DAI), colon length, colonic histopathological structure, and pathological scores. Experimental results show that, compared to the model group, long-chain starch, especially the high-dose group, significantly improved colitis symptoms and reduced colonic tissue damage. Therefore, long-chain starch can serve as a functional product for effectively alleviating colitis. Attached Figure Description Figure 1 The image shows the ion chromatography results of the product obtained under the optimal modification conditions in Example 2.

[0022] Figure 2 The image shows the FTIR results of the product obtained in Example 3.

[0023] Figure 3 This is a diagram showing the chain length distribution at the optimal time for modifying long-branched starch.

[0024] Figure 4 This refers to the animal experiment procedure.

[0025] Figure 5 The DAI scores of mice in each group in Example 4 are shown.

[0026] Figure 6 The colon length of mice in each group in Example 4 is shown.

[0027] Figure 7 The images show H&E staining of colon tissue from mice in each group in Example 4.

[0028] Figure 8 The results show the histopathological scores of the colon tissue of mice in each group in Example 4. Detailed Implementation

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] The culture media involved in the following examples are as follows: LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0031] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0032] Fermentation liquid culture medium: yeast extract 24 g / L, tryptone 12 g / L, KH2PO4 2.32 g / L, K2HPO4 16.44 g / L, glycerol 5 g / L, pH 7.0.

[0033] Detection of long-chain starch First, accurately weigh 50 mg of modified starch sample and dissolve it in 5 mL of dimethyl sulfoxide (DMSO), shaking thoroughly until completely dissolved. Add 1 mL of the above solution to 5 mL of anhydrous ethanol, stir well, and centrifuge at 4000×g for 10 min. Collect the precipitate and redissolve it in 2 mL of 10 mM sodium acetate buffer solution at pH 5. Gelatinize the mixture in a boiling water bath for 10 min, then transfer it to a 50°C constant temperature water bath shaker for 15 min to equilibrate. Add 100 U / mg of isoamylase and react under the same conditions for 24 h. After debranching, terminate the reaction by boiling in a water bath for 30 min. Filter the solution through a 0.22 μm aqueous membrane and analyze the relative proportions of segments with different DP values ​​using HPAEC-PAD.

[0034] Example 1: Expression of recombinant Np-AS enzyme The specific steps are as follows: (1) Strain construction: According to the source Neisseria polysacchareaFor the Np-AS enzyme gene, the upstream primer was designed as 5'-ATGGCCCTGACCCCGAC-3'; the downstream primer was designed as 5'-GTGGTGGTGGTGCTCGAG-3'. Based on the pET-20b(+) vector sequence, the upstream primer was designed as 5'-ATGCTGACCCCGACGCAG-3'; the downstream primer was designed as 5'-CGCAATTTCCAGCCACATCACTTG-3'. The gene and vector were subjected to PCR separately to clone the target gene containing the signal peptide sequence SEQ ID NO.1 (where positions 1-66 are the nucleotide sequence encoding the signal peptide) and the pET20b(+) vector. The PCR system for the Np-AS enzyme gene was as follows: 25 µL of 2×phanta Max Master Mix (Dye plus), 2 µL of forward primer (20 µM), 2 µL of reverse primer (20 µM), 1 µL of template DNA, and double-distilled water was added to a final volume of 50 µL. The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 3 min; followed by 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 3.5 min); and finally, incubation at 72℃ for 5 min. The PCR system for the pET20b(+) vector consisted of 25 µL of 2×phanta Max Master Mix (Dye plus), 2 µL of forward primer (20 µM), 2 µL of reverse primer (20 µM), 1 µL of template DNA, and double-distilled water to a final volume of 50 µL. The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 3 min; followed by 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 4 min); and finally, incubation at 72℃ for 5 min.

[0035] The PCR products of the Np-AS enzyme gene and the pET20b(+) vector were subjected to nucleic acid electrophoresis and then recovered by gel extraction. The Np-AS enzyme gene and the pET20b(+) vector were ligated using homologous recombination. The resulting vector was then transformed into *E. coli* JM109, plated on LB agar plates containing 20 μg / mL ampicillin, and transformed individuals were picked for sequencing and nucleic acid electrophoresis verification to obtain the expression vector pET-20b(+) / Amy containing the Np-AS enzyme gene. The expression vector was then transformed... Escherichia coli BL21(DE3) yielded genetically engineered bacteria. E. coli BL21(DE3)(pET-20b(+) / sp).

[0036] (2) Activation: 100 μL containing genetically engineered bacteria E. coliGlyceryl stock solution of BL21(DE3)(pET-20b(+) / sp) was inoculated into 50 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0) containing 20 μg / mL ampicillin and cultured overnight in a shake flask at 37°C and 200 rpm.

[0037] (3) Fermentation: The seed culture obtained in step (2) was transferred at an inoculum of 4% to 50 mL of TB medium (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 72 mM K2HPO4, pH 6.0) containing 20 μg / mL ampicillin. The medium was then cultured in shake flasks at 25°C and 200 rpm for 6 h. IPTG was then added to a final concentration of 0.05 mM, and the culture was further induced at 25°C (200 r / min) for 36 h. After fermentation, the fermentation broth was centrifuged to collect the cells. The cells were resuspended in 50 mM Tris-HCl (pH 8.0) buffer, sonicated, and the supernatant was collected by high-speed centrifugation. This supernatant was the crude enzyme solution of the recombinant Np-AS enzyme.

[0038] Example 2: Method for preparing long-branched starch The specific steps are as follows: (1) Prepare starch emulsion by adding water to ordinary corn starch, and then stir the starch emulsion at 100 ℃ and 350 rpm for 15 min to mix evenly to obtain gelatinized starch. After gelatinization, place it in a constant temperature water bath at 40 ℃ for 10 min and perform the following (2) or (3) steps respectively. (2) Preparation of modified starch with different enzyme dosages: After the temperature is cooled to room temperature, sucrose of 4 times the weight of starch is added and stirred to dissolve. After being fully dissolved, the starch sucrase Np-AS crude enzyme solution prepared in Example 1 is added to a water bath at 40°C. The enzyme dosages are 0 U / g, 50 U / g, 100 U / g, 150 U / g, 200 U / g, and 250 U / g, respectively. Enzymatic hydrolysis is continued for 16 h. After the hydrolysis is completed, three volumes of anhydrous ethanol are added to the system to terminate the reaction. The product is collected after being repeatedly washed with deionized water and centrifuged three times. (3) Preparation of modified starch with different modification times: After the temperature is cooled to room temperature, add 4 times the mass of starch sucrose and stir to dissolve. After it is fully dissolved, place it in a water bath at 40°C and add 200 U / g of the starch sucrose enzyme Np-AS crude enzyme solution prepared in Example 1. The reaction times are 0 h, 4 h, 8 h, 12 h, 16 h and 20 h respectively. After the reaction is completed, add three times the volume of anhydrous ethanol to the system to terminate the reaction. The product is collected after being washed repeatedly with deionized water and centrifuged three times. (4) The product obtained after enzymatic hydrolysis was placed in a -80℃ freezer and then freeze-dried for 48 h to obtain the modified dry starch. Example 3: Structural characterization of long-branched starch (1) Take 2 mg of the dry starch obtained in Example 2 and grind it evenly with 200 mg of dry KBr powder (spectral grade) in a mortar (particle size < 2 μm, to avoid scattering). Pour the mixture into a tableting mold and press it under vacuum.

[0039] (2) Use the KBr pellet method to quickly transfer the sample to the spectrometer. Set the wavenumber range to 4000-500 cm⁻¹. - ¹, resolution 4 cm - ¹, Scan 16-32 times and take the average.

[0040] Infrared spectroscopy analysis results show that ( Figure 2 The prepared long-chain starch was at 3310.6 cm⁻¹. - The absorption peak of the OH stretching vibration at ¹ and at 1002.3 cm⁻¹ - The CO / COC skeletal vibration absorption peak at ¹ is significantly different from that of ordinary corn starch NCS (the corresponding characteristic absorption peak is located at 3291.8 cm⁻¹). - ¹and approximately 999.4 cm - (¹) All peaks exhibit systematic high wavenumber shifts. These characteristic peak shifts confirm the specificity of the long-branched starch molecule's structure: the shift of the OH absorption peak towards higher wavenumbers indicates enhanced hydrogen bond association between starch molecular chains, forming a more compact hydrogen bond network structure; the shift of the CO / COC vibrational peaks in the fingerprint region further corroborates the rearrangement of sugar chain conformation and the formation of an ordered aggregate structure. Based on this molecular structure, the long-branched starch exhibits significant resistance to enzymatic hydrolysis, thus providing a structural chemical basis for achieving colon-targeted delivery and exerting colitis-relieving effects through intestinal flora fermentation.

[0041] (3) Take 1 g of the dry starch obtained in step (2) of Example 2 and adjust the volume to 10 mL with sodium acetate buffer at pH 4. Add 1 mL of 1000 U / g isoamylase and react at 40 °C and 160 r / min for 24 h to completely debranch the starch. Terminate the reaction by boiling in a water bath for 30 min, then centrifuge at 10000 r / min for 10 min. After diluting 10 times, filter through a 0.22 μm aqueous phase membrane for HPAEC-PAD detection. The results are as follows: Figure 3 As shown, its chain length distribution has changed significantly.

[0042] Table 1. Chain length distribution of ordinary corn starch modified with different enzyme dosages

[0043] The data in the table show that, compared with ordinary corn starch (NCS), the starch chain length distribution shifted significantly after treatment with different enzyme dosages. When the enzyme dosage was 50 U / g and 100 U / g, the degree of starch modification was relatively low, with the products still dominated by medium-sized segments with a degree of polymerization (DP) of 13-24 (accounting for 74.53% and 56.74%, respectively), while the proportion of long branches (DP≥25) was only 12.99% and 40.44%, respectively. This indicates that the enzymatic transglycosylation activity was not fully expressed at these enzyme dosages, and the long chain elongation efficiency was limited. When the enzyme dosage was increased to 150 U / g, the proportion of long branches with DP≥25 increased to 47.26%. When the enzyme dosage was further increased to 200 U / g, the proportion of long branches continued to climb to 54.32%, indicating that under these conditions, the enzymatic transglycosylation reaction dominated the chain length remodeling process. However, when the enzyme dosage was 250 U / g, although the proportion of long-branched starch decreased to 52.46%, the proportion of short-chain starch (DP1~12) also showed an upward trend (reaching 7.85%). In particular, the fluctuation of the monosaccharide components of DP1~5 indicated that the hydrolysis side reaction began to intensify, and excessive enzymatic hydrolysis may lead to a decrease in product purity and yield loss. In summary, choosing an enzyme dosage of 200 U / g can significantly increase the proportion of long-branched starch while effectively inhibiting the excessive formation of short-chain starch and monosaccharides, thereby achieving the targeted and efficient preparation of long-branched starch.

[0044] Example 4: Detection of resistant starch content in modified starch (1) Preparation of mixed enzyme solution: Tube 1: Weigh 4 g of porcine pancreatic amylase and add it to 26.6 mL of distilled water in a 50 mL centrifuge tube. Mix thoroughly and centrifuge at 4000 × g for 10 min. Tube 2: Pipette 1.96 mL of amylase, add 21.0 mg of invertase and 5.04 mL of distilled water, and mix well. Take 34.8 mL of the supernatant from tube 1 and add it to tube 2. Mix well to obtain the mixed enzyme solution.

[0045] (2) Prepare a solution of 50 mg pepsin / 10 mL HCl according to the number of samples. The amount of pepsin is 4 mL × the number of samples. At the same time, add 20 mg of guar gum according to the number of samples and stir well.

[0046] (3) Accurately weigh 200 mg of the dry starch prepared in Example 2 into a 50 mL centrifuge tube, add 2 mL of water, and stir magnetically to ensure that the samples are separated and mixed evenly.

[0047] (4) Place the samples obtained in step (3) in a 37°C water bath shaker. Add 4 mL of pepsin every four minutes. After each sample reacts for 30 min, add six glass beads and 2 mL of sodium acetate buffer. Shake and place in a water bath to continue reacting for 30 min. Inactivate the enzyme in the sample. After inactivation, add 2 mL of the above mixed enzyme to the sample. React accurately for 20 min and 120 min, then take samples to inactivate the enzyme. After inactivation, centrifuge the samples at 10000 r / min for 5 min. Take 50 μL of supernatant, deionized water and standard and mix with 1.5 mL of colorimetric solution (GODPOD kit). React at 37°C for 10 min and measure the absorbance at 520 nm.

[0048] Table 2. In vitro digestion results of modified starch with different enzyme dosages

[0049] Table 3. In vitro digestion results of starch modified at different modification times

[0050] Table 2 shows that the in vitro digestibility of starch was significantly altered after AS enzyme modification. Compared to ordinary corn starch (NCS), the content of rapidly digestible starch (RDS) in all enzyme-added treatment groups showed a significant decreasing trend, while the content of resistant starch (RS) increased substantially. Specifically, although the RS content in the 50 U / g and 100 U / g treatment groups was higher than that in NCS (8.09%), the increase was limited, indicating insufficient modification at these enzyme addition levels. When the enzyme addition was increased to 200 U / g, the RS content rose to 51.40%. Table 3 shows that the effect of modification time on starch digestibility also exhibited a clear pattern. After 16 h of modification treatment, the RS content rapidly increased to 60.41%. Based on the results in Tables 2 and 3, the optimal preparation process was determined to be: enzyme dosage of 200 U / g and modification time of 16 h (the chain length distribution of the product is shown in Table 3). Figure 1 (As shown). Under these conditions, a high conversion rate of resistant starch, a short production cycle, and low enzyme preparation costs can be achieved, enabling the efficient and targeted preparation of long-branched resistant starch.

[0051] Example 5: Application of long-chain amylopectin in alleviating DSS-induced colitis in mice (1) Animal experiments were conducted using long-branched starch prepared with different doses of the optimal process in Example 3 as raw materials: Thirty-two male 6-8 week old C57BL / 6 wild-type mice, weighing 20±2 g, were purchased and housed in an SPF-grade animal facility. Their health met national standards for laboratory animals. The animals were housed in an environment with 25±2 °C and alternating light and dark conditions for 12 hours. They were provided with free access to food and sterile water. The animal experimental procedure is as follows: Figure 4 As shown, C57BL / 6 wild-type mice were randomly divided into four groups of eight mice each: Control group, DSS group, low-dose amylopectin group (100 mg / kg), and high-dose amylopectin group (200 mg / kg). All mice underwent a 7-day acclimatization period before the start of the formal experiment. The intervention period started on day 1 and lasted for 14 days. The treatment methods for each group are as follows: ① Control group: administered physiological saline by gavage daily and drank dd water throughout the treatment period; ② DSS group: administered an equal volume of physiological saline by gavage daily, drank dd water from days 1 to 7, and drank dd water containing 3.0% DSS from days 8 to 14 to establish a colitis model; ③ Low-dose amylopectin group: administered 100 mg / kg amylopectin solution by gavage throughout the treatment period, drank dd water from days 1 to 7, and drank 3.0% DSS solution simultaneously from days 8 to 14 for model establishment; ④ High-dose amylopectin group: administered 200 mg / kg amylopectin solution by gavage throughout the treatment period, drank dd water from days 1 to 7, and drank 3.0% DSS solution simultaneously from days 8 to 14 for colitis model establishment. During the model establishment period, the body weight of mice in each group was measured before each administration, accurate to 0.1 g. Changes in mouse body weight, hair, fecal characteristics, fecal occult blood, and other indicators were observed, and DAI scores were calculated. Mice were sacrificed on day 14 of the intervention period, and the length of the colon was measured. The distal colon was harvested, fixed in 4% neutral formaldehyde, routinely dehydrated, embedded in paraffin, and histological sections were prepared. Some sections were stained with hematoxylin-eosin (HE), and the colonic tissue structure was observed and pathologically scored under a light microscope.

[0052] (2) Experimental method: During the experimental intervention, the weight of mice in each group was measured and recorded daily. Fresh fecal samples were collected daily, and the fecal characteristics were graded and assessed. Fecal occult blood was detected and recorded using a fecal occult blood test kit. The Disease Activity Index (DAI) of mice was assessed using the DAI scoring system. The DAI consists of three scores: degree of weight loss, fecal characteristics, and bleeding. Each score is scored from 0 to 4. The DAI is the average of the three scores: DAI = (weight loss score + fecal score + bleeding score) / 3.

[0053] Table 4 DAI Scoring Criteria

[0054] At the experimental endpoint, mice in each group were euthanized, and colonic tissue was immediately dissected to measure colonic length. The colon was cut along the mesenteric margin and gently rinsed with PBS to remove intestinal contents. The colonic length was measured and recorded after being laid flat. Distal colonic tissue was fixed in 4% neutral formaldehyde fixative, routinely dehydrated, cleared, impregnated with paraffin, and embedded in paraffin to prepare tissue sections. After dewaxing and hydration, the sections were stained with H&E and observed under a light microscope for morphological changes and inflammatory cell infiltration. Quantitative scoring was performed based on pathological scores.

[0055] Table 5. Histopathological scores of mouse colon tissue

[0056] (3) Experimental results: This experiment evaluated the intervention effect of long-chain amylopectin (LCA) on a DSS-induced colitis model in mice. The experiment included a low-dose LCA group (100 mg / kg) and a high-dose LCA group (200 mg / kg), which were compared with the control group and the DSS group. Results showed that LCA improved DSS-induced colitis, demonstrating a clear dose-dependent effect. Regarding the disease activity index (... Figure 5 The DAI score in the DSS model group was significantly higher than that in the blank control group, mainly manifested as abnormal stool characteristics, rectal bleeding, and weight loss, indicating that the enteritis model was successfully established. The DAI score in the high-dose long-chain starch group was significantly lower than that in the DSS model group, and the DAI score in the high-dose group was even lower than that in the low-dose group. Based on this, the colonic tissue inflammation status of each group was further evaluated. The results showed ( Figure 6 The colon length in the DSS group was significantly shorter than that in the control group, exhibiting inflammatory contracture. Both low-dose and high-dose long-chain starch interventions restored colon length to varying degrees, with the high-dose group showing more significant improvement. HE staining showed ( Figure 7 In the control group, the colonic mucosa structure remained intact and the glands were arranged regularly; in the DSS group, significant tissue inflammation and damage were observed, including mucosal defects, gland destruction, reduced goblet cells, inflammatory cell infiltration, and impaired crypt structure. Both low-dose and high-dose long-chain starch interventions alleviated these pathological changes. Figure 8 Further pathological scores confirmed that the DSS group had significantly higher scores than the control group, while the pathological scores of both the low-dose and high-dose long-chain starch groups were lower than those of the DSS group, with the high-dose long-chain starch group showing the most significant improvement. Overall, long-chain starch, especially the high-dose group, can significantly improve colitis symptoms and reduce damage to colonic tissue structure.

[0057] Example 6: An oral formulation An enteric coating comprises 200 mg of amylopectin, 37.5 mg of microcrystalline cellulose, 7.5 mg of hydroxypropyl methylcellulose, and 5 mg of magnesium stearate. The freeze-dried amylopectin is pulverized through an 80-100 mesh sieve, with moisture content controlled to ≤5% to prevent tablet sticking during compression. AOS and microcrystalline cellulose are mixed in equal increments and stirred in a V-type mixer for 15-20 minutes. A binder solution (HPMC dissolved in pure water) is added, and wet granulation is performed, passing the granules through a 16 mesh sieve. The granules are then fluidized bed dried at 50-60℃ until moisture content is ≤3%, granulated, and then mixed with magnesium stearate for 5 minutes. The mixture is then compressed into tablets and finally coated to obtain an oral formulation of amylopectin. The prepared oral formulation has the effect of relieving colitis in mice.

[0058] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A long-branched starch, characterized in that, The chain length distribution percentage of the long amylopectin is as follows: DP1-5, 0.35%; DP6-12, 3.45%; DP13-24, 41.86%; DP25-36, 48.44%; DP≥37, 5.92%.

2. The method for preparing the long-branched starch according to claim 1, characterized in that, Includes the following steps: (1) Pretreatment: Mix starch with water to prepare starch emulsion, heat starch emulsion to gelatinize and cool to room temperature, add three to four times the mass of sucrose, wait for the sucrose to dissolve completely and then place it in a constant temperature water bath at 40℃ for 10 min; (2) Enzymatic hydrolysis: Add starch sucrase Np-AS for 10 to 20 h of enzymatic hydrolysis, and after the end, add three times the volume of anhydrous ethanol to the system to terminate the reaction. The product is collected after washing and centrifugation; (3) Product collection for later use: Freeze the product obtained after enzymatic hydrolysis and then freeze dry it.

3. The method according to claim 2, characterized in that, The amylose sucrase Np-AS is derived from Neisseria polysacchare The amino acid sequence of the starch sucrase Np-AS is shown in SEQ ID NO.

2.

4. The method according to claim 2, characterized in that, The starch mentioned in step (1) is selected from ordinary corn starch.

5. The method according to claim 2, characterized in that, The mass concentration of the starch emulsion in step (1) is at least 10% w / w.

6. The method according to claim 2, characterized in that, In step (2), the amount of starch sucrase Np-AS added is 50~250 U / g dry starch.

7. The use of the long amylopectin of claim 1 in a medicament for relieving or assisting in the improvement of colitis.

8. The application according to claim 7, characterized in that, The dosage forms of the medicine include tablets, capsules, granules, injections, liposome nanoparticles, sustained-release formulations, dispersible tablets, or enteric-coated granules.

9. The application according to claim 8, characterized in that, The drug also contains a pharmaceutical carrier and / or pharmaceutical excipients.

10. An oral formulation containing the long-branched starch of claim 1, characterized in that, The oral formulation contains 75-80% w / w long-chain starch, 17-20% w / w microcrystalline cellulose, and 2-4% w / w magnesium stearate.

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