Application of a safe and effective hippocampal galactolectin 4 short peptide in improving ulcerative colitis

Abstract

This invention discloses the application of a safe and effective hippocampal galactosyllectin 4 short peptide in improving ulcerative colitis. Its amino acid sequence is RCGSDRFKVFLNGQ. This short peptide exhibits high anti-inflammatory activity, significantly alleviating DSS-induced ulcerative colitis symptoms in mice, improving colonic tissue pathological damage, and reducing inflammatory factor expression, with therapeutic effects comparable to mesalazine. The peptide demonstrates good stability, is suitable for oral or enema administration, and exhibits excellent tolerability. Furthermore, toxicological testing confirmed the absence of significant toxic side effects, acute oral toxicity, and risks of gene mutation, chromosomal damage, or aberrations, demonstrating good safety. This invention provides a novel, safe, and highly effective peptide candidate drug for the treatment of ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a safe and effective short peptide of hippocampal galactosyllectin 4 and its application in the preparation of drugs to improve ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease that primarily affects the mucosa of the colon and rectum, characterized by recurrent episodes of inflammation and ulceration. In recent years, with changes in lifestyle and environment, the global incidence of ulcerative colitis has shown a significant upward trend. Patients whose UC is not effectively controlled for a long period may develop colorectal cancer, which has become the third most common malignant tumor worldwide.

[0003] Currently, clinical treatment mainly relies on aminosalicylic acid drugs, glucocorticoids, and immunosuppressants. However, these traditional therapies generally suffer from limited efficacy, significant side effects, and a high relapse rate. In particular, long-term use of glucocorticoids can lead to serious adverse reactions such as osteoporosis and increased risk of infection, while biologics face the dilemma of high treatment costs and secondary failure. Therefore, the development of novel, safe, and effective drugs for the treatment of UC has become an urgent need in the current pharmaceutical field.

[0004] In recent years, peptide drugs have demonstrated unique advantages in the treatment of inflammatory bowel disease due to their high specificity, low toxicity, and good biocompatibility. Compared with traditional small molecule chemical drugs, peptide drugs have the characteristics of strong targeting and safe metabolites. However, existing peptide therapeutics still face technical bottlenecks such as poor intestinal stability and low bioavailability, which seriously limit their clinical application value. How to develop novel therapeutic peptides that combine high efficiency, stability, and safety has become a research hotspot in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a stable, efficient, and safe hippocampal galactosyllectin 4 short peptide that can effectively improve ulcerative colitis in mice.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The amino acid sequence of the short peptide HeGal4 from Hippocampus erectus is: RCGSDRFKVFLNGQ (SEQ ID NO.1).

[0008] This invention has found that the above-mentioned hippocampal galactose lectin 4 has a highly effective anti-inflammatory effect.

[0009] Therefore, the present invention provides the application of the above-mentioned hippocampal galactose lectin 4 in the preparation of anti-inflammatory drugs.

[0010] Preferably, the anti-inflammatory drug also includes pharmaceutically usable excipients.

[0011] Preferably, the anti-inflammatory drug can be formulated into various dosage forms, such as liquid, gas, or solid dosage forms, or powders, tablets, granules, capsules, solutions, emulsions, suspensions, etc.

[0012] Preferably, the drug-resistant agent contains hippocampal galactolectin 4 in a weight fraction of 0.0001-99.9999%, which can be formulated as needed.

[0013] The present invention also provides an anti-inflammatory drug containing the above-mentioned hippocampal galactose lectin 4 as an active ingredient.

[0014] This patented study found that hippocampal galactose lectin-4 has significant anti-inflammatory activity and intestinal protective effects.

[0015] The linear hippocampal Gal4 short peptide provided by this invention can significantly alleviate the symptoms of DSS-induced ulcerative colitis in mice, improve the pathological condition of colonic tissue, and reduce the expression of inflammatory factors. Its therapeutic effect is comparable to that of the marketed positive control drug mesalazine.

[0016] The galactosyllectomycin 4 short peptide of the present invention has good stability and shows good efficacy and good tolerability whether administered orally or via enema.

[0017] Hippocampal galactose lectin 4 exhibits good safety. The polypeptide drug provided by this invention is composed of natural amino acids, has no acute oral toxicity to mice, and does not cause bacterial gene mutations, mammalian erythrocyte chromosome damage, or mammalian cell chromosomal aberrations. Attached image description:

[0018] Figure 1 Oral administration of Gal4 to the hippocampus significantly improved the clinical symptoms of DSS-induced UC in mice, with effects comparable to the positive control drug Ma salazin. * This indicates a significant difference compared to the Ctrl group. # This indicates a significant difference compared to the DSS group.

[0019] Figure 2 Oral administration of Gal4 to the hippocampus significantly improved the pathological condition of colon tissue in DSS-induced mice. * This indicates a significant difference compared to the Ctrl group. # This indicates a significant difference compared to the DSS group.

[0020] Figure 3Oral administration of Gal4 to the hippocampus significantly inhibited the expression of key inflammatory factors in the serum and colon tissue of mice induced by DSS. * This indicates a significant difference compared to the Ctrl group. # This indicates a significant difference compared to the DSS group.

[0021] Figure 4 Seahorse Gal4 enema administration significantly alleviated the apparent symptoms of DSS-induced ulcerative colitis. (A) Fecal occult blood score, (B) Stool formation score, (C) Percentage weight loss, (D) Disease activity index score.

[0022] Figure 5 This is a comparison of the mouse feces at the last sampling point;

[0023] Figure 6 Seahorse Gal4 enema administration significantly improved the histopathological condition of the colon in DSS-induced mice. (A) Display of colon length and condition in mice, (B) Colonic mucosal tissue score, (C) Statistical analysis of colon length data in mice.

[0024] Figure 7 Seahorse Gal4 enema significantly reduced the expression of inflammatory factors in the colon of mice induced by DSS (Mean ± SEM, n = 6). ns indicates no significant difference between groups; ** indicates a significant difference between groups, and p < 0.01. Detailed Implementation

[0025] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0026] Example 1:

[0027] I. Analysis of the Relief Effect of Oral Administration on Colitis

[0028] (1) Oral administration of Gal4 in the hippocampus can significantly alleviate the apparent symptoms of DSS-induced ulcerative colitis.

[0029] The short peptide sequence of the hippocampal Gal4 active domain, RCGSDRFKVFLNGQ, was obtained by chemical synthesis. This short peptide has good solubility and can be dissolved in a variety of solvents. It has good solubility (≤20 mg / mL) in water, 0.1 M PBS (pH 7.4 ± 0.1) and DMSO.

[0030] Male C57BL / 6 mice were provided by Guangzhou University of Chinese Medicine, and the animal experiments were approved by the Ethics Committee of Guangzhou University of Chinese Medicine (No. 20241014020). Healthy mice aged 6 to 8 weeks and weighing between 20 ± 2 g were randomly divided into five groups of 10 mice each. The control group (Ctrl) consisted of healthy mice, and the DSS group was induced by continuous drinking water administration of 2.5% DSS. Gal4-L was administered orally at a low dose of 1 mg / kg, and Gal4-H was administered orally at a high dose of 5 mg / kg twice daily. Mesalazin was used as a positive control group at a dose of 50 mg / kg once daily. Gal4 was administered orally concurrently with DSS drinking water induced modeling for 14 days.

[0031] During the experiment, the mice were weighed regularly and diarrhea, bloody stools, and fecal characteristics were monitored. The Disease Activity Index (DAI) was calculated according to the following formula: DAI = (weight loss score + fecal characteristic score + occult blood score) / 3.

[0032] The disease activity index scoring criteria are as follows:

[0033] Table 1. Standards for Disease Activity Index

[0034]

[0035] The result is shown in the figure. Figure 1 Observation of mouse size showed that the mice in the DSS model group were significantly smaller than those in the control group, while the mice in the Mesalazin group (Mes) and the hippocampus Gal4 administration group were significantly larger than those in the DSS model group. Figure 1 The body weight data in group B showed a significant decrease starting on day 11, indicating successful DSS modeling and the development of inflammatory bowel disease symptoms in the mice. Data in the Mesalazin group fluctuated considerably, but the overall trend was relatively stable, suggesting that Mesalazin had a certain controlling effect on inflammation. Data in the Gal4-L group began to decrease on day 11, but the decrease was less than that in the DSS group, indicating that low-dose Gal4 had a certain alleviating effect on inflammation. Data in the Gal4-L group began to decrease on day 11, but the decrease was less than that in the DSS and Gal4-L groups, indicating that high-dose Gal4 had a more significant alleviating effect on inflammation. High-dose Gal4 had a significant alleviating effect on DSS-induced inflammatory bowel disease, with better efficacy than low-dose Gal4, suggesting that the therapeutic effect of hippocampal Gal4 on enteritis may be positively correlated with dosage. Figure 1The Disease Activity Index (DAI) score was used to assess the severity of the inflammatory bowel disease (IBD) model, combining three indicators: weight loss, stool consistency, and fecal occult blood. The DAI score in the Gal4-L group was higher than that in the Mesalazin group but lower than that in the DSS group, indicating that low-dose Gal4 had some inflammatory-relieving effect, but not as good as Mesalazin. The DAI score in the Gal4-H group was significantly lower than that in the DSS and Gal4-L groups, and slightly lower than that in the Mesalazin group, indicating that high-dose Gal4 had the most significant inflammatory-relieving effect.

[0036] (2) Protective effect of hippocampal Gal4 on colon in DSS-induced UC mouse model

[0037] The colonic mucosal lesions were observed and statistically analyzed using histopathological sections of mouse colon tissue. The scoring criteria were as follows: 0 points for normal colonic tissue with normal epithelial cells and no inflammatory infiltration; 1 point for goblet cell absence and inflammatory infiltration around the crypts; 2 points for extensive goblet cell absence and infiltration of the muscularis mucosa; 3 points for crypt absence, widespread infiltration of the muscularis mucosa, and mucosal thickening; and 4 points for extensive crypt absence and severe inflammatory infiltration of the submucosa. Figure 2 In mice in groups A and B, the colon length was significantly shortened in the DSS group, while the colon length in the Gal4-L group recovered slightly. The colon lengths in the Gal4-H and Mesalazin groups were comparable, with both showing significant recovery. Figure 2 C. Healthy mice showed intact and healthy colon tissue, while the DSS group mice showed obvious erosion, inflammation, and ulceration in their colon tissue. The Gal4-L group showed slight improvement, while the Gal4-H group and Mesalazin showed the best improvement.

[0038] (3) Effects of hippocampal Gal4 on inflammatory factors in serum and colon tissue of DSS-induced mice

[0039] Serum and colon tissue samples were collected from mice on day 14, and cytokine levels were detected by ELISA, mainly including IL-1β, IL-6, IFN-γ, and TNF-α. The results are as follows: Figure 3 As shown, the expression patterns of the above four cytokines in serum and colon are very similar. The expression levels of these cytokines in the DSS group were significantly higher than those in the control group, while the expression levels in the Gal4-L group were slightly decreased. The expression levels in the Gal4-H and Mesalazin groups were significantly decreased, with similar degrees of decrease in both groups. The therapeutic effect of high-dose Gal4 and the positive control drug Mesalazin indicate that hippocampal Gal4 has a highly effective anti-enteritis effect.

[0040] II. Analysis of the Relief Effect of Enema Administration on Colitis

[0041] (1) Gal4 in the hippocampus can significantly alleviate the apparent symptoms of DSS-induced ulcerative colitis.

[0042] Healthy mice aged 6 to 8 weeks and weighing between 20 ± 3 grams were randomly divided into four groups of 8 mice each. The Saline group received normal drinking water and was given an enema with saline solution from day 7 to 16. The Saline + Gal4 group received normal drinking water and was given an enema with Gal4 peptide dissolved in saline solution from day 7 to 16, once daily at a dose of 5 mg / kg. The DSS group served as the model group, receiving 5% DSS in drinking water from day 1 to 6 until successful model establishment. From day 7 to 16, DSS administration was discontinued, and mice were given an enema with saline solution once daily. The DSS + Gal4 peptide group received DSS in drinking water from day 1 to 6 and was given an enema with Gal4 peptide dissolved in saline solution from day 7 to 16, once daily at a dose of 5 mg / kg.

[0043] The results are as follows Figure 4 As shown, mice exhibited bloody stools on day 4 of DSS induction, reaching their most severe state of bloody stools and diarrhea on day 6, with almost no appetite and extremely depressed condition, indicating successful establishment of the mouse colitis model. DSS administration was discontinued on day 7, followed by rectal injection of hippocampal Gal4 peptide. On the first day after administration, bloody stools, fecal indices, and weight loss began to improve. With increasing administration days, the alleviating effect of hippocampal Gal4 on mouse enteritis became increasingly significant, showing a significant difference between the treatment group and the model group on day 8. At the final sampling, the weight, fecal condition, and overall health of both the saline group and the saline+Gal4 group were within healthy ranges, with no significant differences between the two groups. The DSS model group had the most severe disease index and severe diarrhea, while the Gal4 group showed the best fecal condition. Figure 5 As shown. In conclusion, hippocampal Gal4 can significantly alleviate the symptoms of DSS-induced ulcerative colitis.

[0044] (2) Gal4 in the hippocampus can significantly alleviate the apparent symptoms of DSS-induced ulcerative colitis.

[0045] In mice not fed DSS, there was no significant difference in colon length between the Saline and Saline+Gal4 groups, and the feces in the colon were granular, indicating a very healthy colon. Figure 6 A). The colonic mucosal condition (erosion, inflammation, and ulceration) of mice not induced by DSS was good, and there was no significant difference between the Saline and Saline+Gal4 groups. Figure 6 B). This indicates that Gal4 in the hippocampus does not damage the health of the colonic mucosa in mice.

[0046] The colon length of mice induced by DSS was atrophied, while the colon length atrophy was significantly alleviated in mice treated with hippocampal Gal4 peptide. Figure 6 (A and C). Compared with the DSS group, although there was no significant difference in the scores of the colonic mucosa between the DSS+Gal4 group and the DSS group, the symptoms of colonic erosion, inflammation, and ulceration were slightly alleviated. Figure 6 B).

[0047] (3) Gal4 in the hippocampus significantly reduced the expression of inflammatory factors in DSS-induced mouse colon tissue.

[0048] Mouse colon tissue samples were collected, and the expression levels of inflammatory factors in the DSS group and the DSS+Gal4 group were detected using qRT-PCR. Results are as follows: Figure 7 The results showed no significant differences in IL-6 and IL-10 between the two groups. However, TNF-α, IL-1β, IL-8, and IL-17 were significantly downregulated in the DSS+Gal4 group, indicating that hippocampal Gal4 peptide can significantly alleviate DSS-induced colonic inflammation.

[0049] III. Toxicological Studies of Hippocampal Galactolectin 4 (Gal4)

[0050] We commissioned the Health Testing Center of Southern Medical University in Jiangsu Province to conduct acute oral toxicity analysis, bacterial reverse mutation analysis, mammalian erythrocyte micronucleus analysis, and in vitro mammalian cell chromosome aberration analysis on the seahorse Gal4 peptide.

[0051] (1) Acute oral toxicity analysis of hippocampal galactose lectin 4 (Gal4)

[0052] 1. Materials and Methods

[0053] 1.1 Sample:

[0054] The amino acid sequence of hippocampal Gal4 peptide (Hegal-4) is RCGSDRFKVFLNGQ, provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences, and cryopreserved. Take 0.0050 g of the sample, add purified water to 5 mL to achieve a concentration of 1 mg / mL, and mix thoroughly to obtain the test substance.

[0055] 1.2 Experimental animals:

[0056] Twenty healthy adult SPF-grade ICR mice, with 10 males and 10 females, weighing 19.0 - 20.9 g for females and 19.3 - 21.6 g for males, were provided by Nanjing Medical University, with the production license number SCXK(Su)2021-0001 and the certificate number: A202408160028. The experimental animals were housed in the central barrier system at a temperature of 20 - 26°C and a relative humidity of 30% - 70%. The experimental animal use license number is SYXK(Su)2020-0006. The maintenance feed for the experimental mice was provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., with the production license: Su Feed License (2019)01008.

[0057] 1.3 Dose selection and administration method of the test substance:

[0058] The limit test method was used for the acute oral toxicity test of mice. The animals were quarantined for 4 days, the dose was 10000 mg / kg·bw, the specific gravity of the test substance was 1.00 g / mL, the animals were fasted for 4 h before the test, the test substance was given as the stock solution, the gavage volume was 10 mL / kg, and the sample was given once, with free access to water.

[0059] The animals were continuously observed for 14 days after giving the sample, and the toxic manifestations and death conditions were recorded. The body weights were measured on the 0th day, 7th day, and 14th day. At the end of the observation period, the animals were subjected to gross anatomical examination.

[0060] 1.4 Main instruments: ME104E / 02 electronic balance, JJ1000 electronic balance.

[0061] 1.5 Test method: The limit test method was used for the acute toxicity test of mice.

[0062] 1.6 Result determination: The test substance was classified according to the acute toxicity dose classification table.

[0063] Table 2 Acute toxicity (LD50) dose classification table

[0064]

[0065] 2 Results

[0066] [[ID=第30]]During the test observation period, the animals had normal diet and activities, grew well, showed no toxic manifestations or deaths, and no abnormal pathological changes were observed during the gross anatomical examination of the animals at the end of the observation period. The effects of Hegal-4 (solution of 1 mg / mL) on the body weight and death number of mice are shown in Table 3.

[0067] ​​​​​​​3. Summary

[0070] No obvious signs of poisoning were observed after oral administration of the test product to animals. It had no significant effect on animal weight gain, and no animals died during the observation period. The acute oral toxicity test of a 1 mg / mL solution of Hegal-4 in male and female mice yielded [LD50]. 50 All values ​​were greater than 10000 mg / kg·bw. According to the National Food Safety Standard GB 15193.3-2014, the acute toxicity (LD50) is... 50 According to the dose grading table, this sample is practically non-toxic.

[0071] (2) Effects of hippocampal galactolectin 4 (Gal4) on bacterial reverse mutation

[0072] 1. Materials

[0073] 1.1 Sample

[0074] Hegal-4, produced by Jiangsu Genscript Biotech Co., Ltd. and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences, was used for sample cryopreservation. When using it, it was dissolved in purified water to a concentration of 1 mg / mL. The solution after thorough dissolution and mixing was used as the test sample.

[0075] 1.2 Experimental strains and activation system

[0076] 1.2.1 Strains

[0077] The Salmonella Typhimurium mutant strains TA97a, TA98, TA100, TA102 and TA1535 were all derived from Molecular Toxicology, Inc.

[0078] 1.2.2 Activation System

[0079] The activation system was rat liver S9 induced by β-naphthylflavonoid and phenobarbital sodium. The S9 protein content was 30 mg / mL, and its activity showed good metabolic activation ability for 2-aminofluorene. S9 was obtained from Jiangsu Qishi Biotechnology Co., Ltd., batch number: 24FS008E.

[0080] 1.3 Main Reagents and Instruments

[0081] 1.3.1 Main Reagents

[0082] 2-Aminofluorene, sourced from Aladdin, batch number: J2116394; 1,8-dihydroxyanthraquinone, sourced from Aladdin, batch number: G2208134; 2,4,7-trinitrofluorenone, sourced from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd., batch number: 1380705CY; methyl methanesulfonate, sourced from Aladdin, batch number: K2216288; sodium azide, sourced from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd., batch number: 87Q2; cyclophosphamide, sourced from Baxter Oncology GmbH, batch number: 3C595A.

[0083] 1.3.2 Main Instruments

[0084] JJ1000 electronic balance, AL104 electronic balance, biosafety cabinet, electric thermostatic incubator, autoclave, THZ-312 thermostatic shaker, digital display thermostatic water bath, Millipore pure water system.

[0085] 2 methods

[0086] According to the requirements of the bacterial reverse mutation test in the national food safety standard GB 15193.4—2014, mutant strains of Salmonella Typhimurium, TA97a, TA98, TA100, TA102, and TA1535, were selected and tested after passing the identification. The first test used the plate incorporation method to confirm the use of the pre-cultured plate incorporation method.

[0087] 2.1 Solvent, Dosage

[0088] 2.1.1 Solvent

[0089] Purified water was selected as the solvent for the test samples in this experiment. DMSO was used as the solvent for the positive controls 2-aminofluorene, 1,8-dihydroxyanthraquinone, 2,4,7-trinitrofluorenone, and methyl methanesulfonate, while purified water was used as the solvent for sodium azide and cyclophosphamide.

[0090] 2.1.2 Negative control group

[0091] All experiments (initial experiments and confirmatory experiments) must include an untreated control group and a solvent control group (purified water, DMSO).

[0092] 2.1.3 Test Dosage

[0093] Initial test: Doses were set at 5000 μg / plate, 1581 μg / plate, 500 μg / plate, 158.1 μg / plate, and 50 μg / plate. The sample was prepared into a 50000 μg / mL solution using purified water, and then serially diluted with purified water to prepare four concentrations: 15810 μg / mL, 5000 μg / mL, 1581 μg / mL, and 500 μg / mL. 100 μL of each solution was added to each plate before use. This initial test was used to determine the solubility and bacterial toxicity of the test substance.

[0094] Confirmatory test: Based on the results of the initial test, the doses for the confirmatory test were set at 5000 μg / plate, 1000 μg / plate, 200 μg / plate, 40 μg / plate and 8 μg / plate. The samples were prepared into a solution with a concentration of 50000 μg / mL using purified water, and then further diluted with purified water at certain ratios to prepare solutions with four concentrations of 10000 μg / mL, 2000 μg / mL, 400 μg / mL and 80 μg / mL. 100 μL of each solution was added to each plate before use.

[0095] 2.1.4 Positive control group

[0096] Positive controls are selected based on the type of bacterial strain, as follows:

[0097] Table 4 Positive Controls

[0098]

[0099] 2.2 Operating Procedures

[0100] For the plate incorporation method, add 0.1 mL of enrichment broth for the test strain, 0.1 mL of the test sample solution, and 0.5 mL of S9 mixture (add 0.5 mL of phosphate buffer if S9 activation is not required) to 2 mL of top agar. After mixing, pour the mixture onto bottom agar plates, three plates per dose per strain, and incubate at 37°C for 48 h. Count the number of revertant colonies per plate.

[0101] For the pre-culture plate incorporation method, the following pre-culture steps are performed before adding the top agar: In the experiment, the test sample (add 10% S9 mixture if activation is required) and bacterial suspension are incubated at 37℃ for 20 min, and then 2 mL of top agar is added and quickly poured onto the bottom culture medium. The plate is rotated to distribute the top culture medium evenly on the bottom, laid flat to solidify, and incubated at 37℃ for 48 h. The number of revertant colonies in each plate is counted.

[0102] 2.3 Data Processing and Result Evaluation

[0103] 2.3.1 Data Processing

[0104] Calculate the mean and standard deviation of the number of reverted colonies on three plates for each strain and each dose.

[0105] 2.3.2 Results Evaluation

[0106] Under favorable background growth conditions, a positive result can be determined if the number of revertant colonies of test strains TA98 and TA1535 is equal to or greater than twice that of the untreated control group, and the number of revertant colonies of other test strains is equal to or greater than twice that of the untreated control group, and one of the following two conditions (a) or (b):

[0107] a) A dose-response relationship exists;

[0108] b) A test point has a repeatable positive result.

[0109] 3 Results

[0110] 3.1 Sterility test results

[0111] No contaminated colonies were observed on sterile plates for the test sample solution, solvent control, buffer solution, and S9 mixture.

[0112] 3.2 Results of bacterial reverse mutation test

[0113] Initial test results showed that no precipitation was observed in any dose group of the test samples under both metabolic activation and non-metabolic activation conditions, and no significant toxicity of the test samples to the test strains was observed in any dose group.

[0114] The experimental results confirmed that no precipitation was observed in any dose group of the test samples under both metabolic activation and non-metabolic activation conditions, and no significant toxicity of the test samples to the test strains was observed in any dose group.

[0115] As shown in Tables 5 and 6, the number of revertant colonies of strains TA97a, TA98, TA100, TA102, and TA1535 in each dose group of the tested samples was less than twice that of the control group, and there was no dose-response relationship. Therefore, the bacterial reversion mutation test results of hippocampal Gal4 peptide were negative in both cases with and without S9.

[0116] Table 5. Results of bacterial reversion mutation test colony count (initial test, plate incorporation method)

[0117]

[0118]

[0119] Note: "-" indicates that counting is affected by precipitation. "a" indicates more than twice the number of colonies in the untreated control group. Classification of precipitation: A hazy feeling caused by fine particles indicates no precipitation observed; non-interfering precipitation means that precipitation can be seen with the naked eye on the plate, but the precipitation particles are less than or equal to 10% of the number of revertant colonies; interfering precipitation means that precipitation can be seen with the naked eye on the plate, but the precipitation particles exceed 10% of the number of revertant colonies, and these plates must be counted manually; severely interfering precipitation means that precipitation affects colony counting and observation of the background bacterial lawn, and this data should be discarded.

[0120] Table 6 Results of counting the number of revertant colonies in the bacterial reverse mutation test (confirmation test, pre-incubation plate incorporation method)

[0121]

[0122]

[0123] Note: "a" indicates more than twice the number of colonies in the untreated control group. Classification of precipitation: A hazy feeling caused by fine particles indicates no precipitation observed; non-interfering precipitation means that precipitation can be seen with the naked eye on the plate, but the precipitation particles are less than or equal to 10% of the number of revertant colonies; interfering precipitation means that precipitation can be seen with the naked eye on the plate, but the precipitation particles exceed 10% of the number of revertant colonies, and these plates must be counted manually; severely interfering precipitation means that precipitation affects colony counting and observation of the background bacterial lawn, and this data should be discarded.

[0124] (3) Effect of hippocampal galectin-4 (Gal4) on micronuclei in mammalian red blood cells

[0125] 1 Materials

[0126] 1.1 Samples

[0127] Hegal-4, produced by Jiangsu GenScript Biotech Co., Ltd. and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. The samples were stored frozen and dissolved in purified water before use to a concentration of 1 mg / mL. The solution after full dissolution and mixing was the test sample.

[0128] 1.2 Experimental animals

[0129] SPF-grade ICR mice, provided by Nanjing Medical University, production license number: SCXK (Jiangsu) 2021-0001, certificate number: A202410220610.

[0130] 1.3 Main reagents and instruments

[0131] 1.3. Main reagents

[0132] Cyclophosphamide, sourced from Baxter Oncology GmbH, batch number: 3C595A.

[0133] Normal saline, sourced from Jiangsu Huaian Double Crane Pharmaceutical Co., Ltd., batch number: 2310263.

[0134] Methanol, sourced from China National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 20240326.

[0135] Fetal bovine serum, sourced from Zhejiang Tianhang Biotechnology Co., Ltd., batch number: 22050201.

[0136] Giemsa, sourced from Beyotime, batch number: 081623240207.

[0137] 1.3.2 Main instruments

[0138] JJ1000 electronic balance, JJ100 electronic balance, AL104 electronic balance, OLYMPUS microscope.

[0139] 1.4 Rearing conditions

[0140] The experimental animals were reared in a barrier system with a temperature of 20.0°C to 26.0°C and a relative humidity of 3***********icense number for the use of experimental animals: SYXK (Jiangsu) 2020 - 0006. The irradiated sterilized feed was provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., production license: Su Feed License (2014) 01008.

[0141] 2 Methods

[0142] According to the requirements of the national food safety standard "Mammalian erythrocyte micronucleus test" (GB 15193.5—2014), 50 ICR mice weighing 25 - 35 g were selected. After the animals were purchased, they were allowed to acclimatize to the environment for 3 days before the experiment. They were randomly divided into 5 groups of 10 each, with an equal number of males and females. The 30 - hour administration of the test substance method was used.

[0143] 2.1 Solvent, dosage

[0144] 2.1.1 Solvent

[0145] Purified water was selected as the solvent for the test samples in this experiment.

[0146] 2.1.2 Dosage

[0147] The high dose was set at 10000 mg / kg·bw, and the medium and low doses were 5000 and 2500 mg / kg·bw respectively. There were 3 dose groups, and a solvent control group (purified water) was also set. They were gavaged at 20 mL / kg·bw; a positive control group (cyclophosphamide, intraperitoneal injection at 40 mg / kg·bw). See the following table for details:

[0148]

[0149]

[0150] 2.2 Operating Procedures

[0151] The 30-hour administration method was used, with two administrations of the test sample 24 hours apart. Animals were sacrificed 6 hours after the second administration, and sternal sections were routinely prepared and examined microscopically. 2000 polychromatic erythrocytes (PCEs) were counted per mouse, and the number of polychromatic erythrocytes containing micronuclei was observed and the micronucleus incidence rate was calculated as a percentage per thousand. The number of mature red blood cells (NCEs) observed when 200 polychromatic erythrocytes were counted per mouse was also calculated, and the ratio of polychromatic erythrocytes to total red blood cells (PCE / (PCE+NCE)) was calculated.

[0152] 2.3 Data Processing and Result Evaluation

[0153] 2.3.1 Data Processing

[0154] The mean and standard deviation of micronucleated cell rate were calculated separately for each group according to animal sex. The micronucleated cell rate of each dose group of the test sample was compared with that of the solvent control group, and the U test was used for statistical analysis.

[0155] 2.3.2 Results Evaluation

[0156] A positive result is confirmed when the experimental group shows a significant dose-response relationship and statistical significance in the micronucleated cell rate compared to the control group. If the difference is statistically significant but there is no dose-response relationship, the test should be repeated. A positive result is confirmed if the result can be replicated.

[0157] 3 Results

[0158] As shown in Table 7, there was no significant difference in the micronucleus rate between the test sample groups and the negative control group (P>0.05), while the positive control group showed a significantly higher rate than the negative control group (P<0.05). The PCE / (PCE+NCE) ratio of each test sample group was less than 20% different from the control group, indicating that the test sample did not inhibit bone marrow cell proliferation. No effect was observed on micronucleus formation in mouse bone marrow polychromatic erythrocytes or on the polychromatic erythrocyte to total erythrocyte ratio (PCE / (PCE+NCE)). Therefore, the Hegal-4 mammalian erythrocyte micronucleus test result was negative.

[0159] Table 7. Effects of the tested samples on the micronucleus incidence and the ratio of polychromatic erythrocytes to total erythrocytes (PCE / (PCE+NCE)) in mouse bone marrow cells.

[0160]

[0161] Note: *Compared with the negative control group, P<0.05.

[0162] (4) Effects of hippocampal galactolectin 4 (Gal4) on chromosomal aberrations in mammalian cells in vitro

[0163] 1. Materials

[0164] 1.1 Sample

[0165] Hegal-4, produced by Jiangsu Genscript Biotech Co., Ltd. and provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences, was used for sample cryopreservation. When using it, it was dissolved in purified water to a concentration of 1 mg / mL. The solution after thorough dissolution and mixing was used as the test sample.

[0166] 1.2 Experimental cell lines and activation system

[0167] 1.2.1 Cell lines

[0168] The Chinese hamster lung (CHL) cell line was obtained from the cell bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0169] 1.2.2 Activation System

[0170] The activation system was rat liver S9 induced by β-naphthylflavonoid and phenobarbital sodium. The S9 protein content was 30 mg / mL, and its activity showed good metabolic activation ability for cyclophosphamide. S9 was obtained from Jiangsu Qishi Biotechnology Co., Ltd., batch number: 23FS037K.

[0171] 1.3 Main Reagents and Instruments

[0172] 1.3.1 Main Reagents

[0173] Cyclophosphamide, sourced from Baxier Oncology GmbH, batch number: 3C595A.

[0174] Mitomycin, sourced from Saen Chemical Technology Co., Ltd., batch number: 93SEROFX.

[0175] MEM culture medium, sourced from Biosharp, batch number: 23348168.

[0176] Fetal bovine serum, sourced from Zhejiang Tianhang Biotechnology Co., Ltd., batch number: 24020704.

[0177] 0.25% trypsin solution, sourced from Gibco, batch number: 2661767.

[0178] Colchicine, sourced from Tokyo Seika Kogyo Co., Ltd., batch number: 6ECLG-KQ.

[0179] Giemsa staining solution, sourced from Beyotime Biotechnology, batch number: 081623240207.

[0180] 1.3.2 Main Instruments

[0181] JJ100 electronic balance, AL104 electronic balance, OLYMPUS microscope, clean bench, CO2 incubator, autoclave.

[0182] 2 methods

[0183] In accordance with the requirements of GB 15193.23—2014 National Food Safety Standard for In Vitro Mammalian Cell Chromosomal Aberration Test, the Chinese hamster lung (CHL) cell line was selected.

[0184] 2.1 Solvent, Dosage

[0185] 2.1.1 Solvent

[0186] In this experiment, serum-free MEM culture medium was selected as the solvent for the test samples.

[0187] 2.1.2 Negative control group

[0188] All experiments (preliminary and formal experiments) must include a solvent control group (serum-free MEM culture medium).

[0189] 2.1.3 Test Dosage

[0190] Based on cytotoxicity and sample solubility, three dosage groups of 5000 μg / mL, 2500 μg / mL, and 1250 μg / mL were selected for chromosomal aberration testing.

[0191] 2.1.4 Positive control group

[0192] Under conditions without metabolic activation, mitomycin C, a direct mutagen, was used as a positive control at a concentration of 0.25 μg / mL; under conditions with metabolic activation, cyclophosphamide, an indirect mutagen, was used as a positive control at a concentration of 15 μg / mL.

[0193] 2.2 Operating Procedures

[0194] The day before the experiment, a certain number of cells were seeded in culture dishes and incubated in a CO2 incubator. During the experiment, the culture medium was aspirated from the dishes, and a certain concentration of the test substance, S9 mix (if S9 mix was not added, it needed to be supplemented with culture medium), and a certain amount of serum-free culture medium were added. The dishes were then incubated for 3 hours (in addition to the 3-hour treatment without S9 mix, a separate 24-hour treatment was performed). After treatment, the culture medium containing the test substance was aspirated, the cells were washed three times with Hanks' solution, and culture medium containing 10% fetal bovine serum was added. The cells were returned to the incubator, and harvested after 24 hours. Four hours before harvest, colchicine, a cell division metaphase inhibitor, was added for 4 hours at a final concentration of 1 μg / mL. The harvested cells were digested, hypotonic, fixed, and then slided and stained for slide review. Slides were reviewed under an oil immersion microscope, and at least 100 well-dispersed metaphase cells were analyzed for each dose group.

[0195] 2.3 Data Processing and Result Evaluation

[0196] 2.3.1 Data Processing

[0197] Data are listed by dosage, and indicators include the number of observed cells, the number of aberrant cells, the chromosomal aberration rate, and the number and rate of different types of chromosomal aberrations in each dosage group and control group. Clefts should be recorded and reported separately, but are generally not included in the total aberration rate. The chromosomal aberration rate for each group is expressed as χ². 2 The test results were statistically analyzed.

[0198] 2.3.2 Results Evaluation

[0199] The following two conditions can be used to determine that the test sample is a positive result in this test: 1) The increase in the number of chromosomal structural aberrations caused by the test sample is statistically significant and dose-related; 2) The increase in the number of chromosomal structural aberrations caused by the test sample under any dose condition is statistically significant and reproducible.

[0200] 3 Results

[0201] 3.1 Results of Cytotoxicity Assay (Preliminary Assay)

[0202] As shown in Table 8, no cytotoxicity was observed in any of the dosage groups. No precipitation was observed in any dosage group after sample addition; no precipitation was observed in any dosage group after 3 hours of incubation; and no precipitation was observed in any dosage group after 24 hours of incubation. Based on cytotoxicity and sample solubility, three dosage groups (5000 μg / mL, 2500 μg / mL, and 1250 μg / mL) were selected for the chromosomal aberration assay.

[0203] 3.2 Results of Chromosomal Aberrations

[0204] The solubility of the test substance, pH value, and results of the chromosome aberration test for each dose group are shown in Tables 9 and 10.

[0205] Under conditions of no metabolic activation treatment for 3 hours, the chromosomal aberration rate of cells in each dose group of the test samples was less than 5%. Compared with the solvent control group, the rate of no chromosomal aberrations in each dose group was significantly increased (P>0.05, χ²). 2 (Test). The chromosomal structural aberration rate in the positive control group (mitomycin C) was 21.0%, which was statistically significantly increased compared with the solvent control group (P<0.05, χ²). 2 test).

[0206] Under metabolic activation treatment for 3 hours, the chromosomal aberration rate of cells in each dose group of the test samples was less than 5%. Compared with the solvent control group, the rate of no chromosomal aberrations in each dose group was significantly increased (P>0.05, χ²). 2 (Test). The chromosomal structural aberration rate in the positive control group (cyclophosphamide) was 16.0%, which was statistically significantly increased compared with the solvent control group (P<0.05, χ²). 2 test).

[0207] Under conditions of no metabolic activation treatment for 24 hours, the chromosomal aberration rate of cells in each dose group of the test samples was less than 5%. Compared with the solvent control group, the rate of no chromosomal aberrations in each dose group was significantly increased (P>0.05, χ²). 2 (Test). The chromosomal structural aberration rate in the positive control group (mitomycin C) was 23.0%, which was statistically significantly increased compared with the solvent control group (P<0.05, χ²). 2 test).

[0208] Therefore, the Hegal-4 in vitro mammalian cell chromosome aberration test result was negative.

[0209] Table 8 Cytotoxicity assay

[0210]

[0211]

[0212] Rating criteria: ++ = After the test substance solution is added to the culture medium solution, the test substance precipitates out in large quantities, forming a large amount of precipitate; + = After the test substance solution is added to the culture medium solution, the test substance precipitates out in small quantities, forming a small amount of precipitate; — = Completely dissolved.

[0213] Table 9 Summary of Chromosomal Aberration Test Results

[0214]

[0215]

[0216] Note: *P<0.05 compared with the negative control group; rating criteria: ++=The test substance precipitates out in large quantities after the test substance solution is added to the culture medium solution; +=The test substance precipitates out in small quantities after the test substance solution is added to the culture medium solution; —=Completely dissolved; treatment time: refers to the contact time between the test substance and the cells.

[0217] Table 10 Results of Chromosomal Aberration Tests

[0218]

[0219]

[0220]

Claims

1. Application of hippocampal galactolectin 4 in the preparation of anti-ulcerative colitis drugs, wherein the amino acid sequence of hippocampal galactolectin 4 is: RCGSDRFKVFLNGQ.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically usable excipients.

3. The application according to claim 1, characterized in that, The drug is in liquid, gas, or solid dosage form.

4. The application according to claim 1, characterized in that, The drugs mentioned are powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

5. The application according to claim 1, characterized in that, The drug contains hippocampal galactosyllectrin 4, which has a weight fraction of 0.0001-99.9999%.

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