Use of a natural oyster zinc peptide product in the preparation of a medicine for preventing and treating intestinal inflammation

Abstract

The application discloses application of a natural oyster zinc peptide product in preparation of a medicine for preventing and treating enteritis. The application verifies the function of the natural oyster zinc peptide product in preventing and treating enteritis through animal experiments, the natural oyster zinc peptide product can improve the apparent symptoms of ulcerative colitis of mice caused by DSS by regulating the oxidation stress level, relieving the inflammatory reaction, maintaining the intestinal barrier and reducing the cell apoptosis, and the natural oyster zinc peptide product can also regulate the intestinal flora and metabolic products of the intestinal flora of the enteritis mice, so that the intestinal inflammation is relieved. The natural oyster zinc peptide product used in the application has good safety, compared with common anti-inflammatory and immunity-enhancing medicines, the natural oyster zinc peptide product has lower cost and no side effect, and the effect of relieving enteritis is remarkable. Therefore, the natural oyster zinc peptide product has a good prospect in development of medicines for preventing and treating enteritis.

Description

Technical Field

[0001] This invention belongs to the field of food and health care, and specifically relates to the application of a natural oyster zinc peptide product in the preparation of drugs for the prevention and treatment of enteritis. Background Technology

[0002] In recent years, inflammatory bowel disease (IBD) has become one of the most serious chronic diseases threatening human health. Symptoms of IBD typically include inflammation or ulceration of the colon or rectum, diarrhea, rectal bleeding, and weight loss, with a prolonged course. Ulcerative colitis (UC) relapses are frequently accompanied by complications, severely impacting patients' quality of life. Research data shows that the incidence of UC is increasing year by year, especially in developed countries. However, medications for treating UC are often expensive and have many side effects, such as acne, abdominal pain, weight gain, and diabetes. Therefore, it is imperative to find functional components from food-derived bioactive substances to treat or alleviate UC.

[0003] To explore the potential pathogenesis of IBD and identify new therapeutic targets, various animal models have been used in experiments. Among them, the DSS-induced UC animal model is the most commonly used. By adding DSS to drinking water, animals can develop UC-like symptoms, making it one of the ideal animal models for studying the therapeutic effects of IBD. DSS has toxic effects on colonic epithelial cells, disrupting the intestinal barrier, leading to increased intestinal permeability, promoting the entry of inflammatory cytokines and toxins through the intestinal wall, activating related inflammatory signaling pathways, and causing inflammatory damage. This results in experimental animals exhibiting significant symptoms such as weight loss, diarrhea, bloody stools, and reduced activity. This method is convenient to operate, highly reproducible, and widely used in enteritis models.

[0004] A growing body of research indicates that the beneficial effects of numerous food components and nutrients, such as proteins and bioactive peptides, on enteritis-associated ulcerative colitis (UC) far exceed their traditional nutritional value. Proteins and other nutrients serve as carbon and nitrogen sources for gut microbiota growth, and their metabolism generates SCFAs, which participate in the regulation of inflammatory responses. The functional studies of oyster bioactive peptides are increasingly rich, revealing their ability to improve zinc ion bioavailability, as zinc possesses certain anti-inflammatory activity. The natural oyster zinc peptide product prepared in this invention is derived from Pacific oysters, a readily available raw material with good safety and no side effects, thus showing great potential in the development of drugs for the prevention and treatment of enteritis. Summary of the Invention

[0005] This invention provides the application of a natural oyster zinc peptide product in the preparation of a drug for the prevention and treatment of enteritis. The natural oyster zinc peptide product is abundant and readily available, has good safety profile, and exhibits significant effects in improving enteritis. It can also regulate the intestinal flora and its metabolites in mice with enteritis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the application of a natural oyster zinc peptide product in the preparation of a drug for the prevention and treatment of enteritis.

[0008] Furthermore, the natural oyster zinc peptide product contains four peptide segments with good binding ability to zinc ions; the amino acid sequences of the peptide segments are: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT, and SGPSIVH.

[0009] Furthermore, the preparation method of the natural oyster zinc peptide product is as follows:

[0010] (1) Remove the shells from the oysters, clean the oyster meat, add 2 to 4 times the volume of deionized water and crush to obtain the crushed tissue fluid;

[0011] (2) The tissue homogenate from step (1) is subjected to ultrasonic disruption, and the resulting tissue homogenate is centrifuged to collect the supernatant.

[0012] (3) The supernatant from step (2) was rotary evaporated and freeze-dried to obtain crude natural oyster zinc peptide product;

[0013] (4) The crude natural oyster zinc peptide product from step (3) is separated and purified to obtain the natural oyster zinc peptide product.

[0014] Furthermore, in step (2), the ultrasonic breaking power is 70 W - 100 W, and the time is 15 min - 30 min.

[0015] Furthermore, the effective dose of the natural oyster zinc peptide product is 1.25 g / kg / bw to 5 g / kg / bw.

[0016] Furthermore, the natural oyster zinc peptide product can significantly reduce weight loss in mice caused by DSS, improve abnormal fecal condition, and inhibit the increase of DAI score in mice.

[0017] Furthermore, the natural oyster zinc peptide product can alleviate the superficial and internal tissue lesions of the colon induced by DSS, maintain the integrity of the colon tissue in mice, and play a positive role in stabilizing the intestinal environment.

[0018] Furthermore, the natural oyster zinc peptide product can prevent and treat enteritis by downregulating the expression of pro-inflammatory factors, mediating the activation of the NF-κB pathway, regulating the structure of intestinal flora, and increasing the content of SCFAs.

[0019] Furthermore, the natural oyster zinc peptide product can downregulate the expression of pro-inflammatory factors IL-6, TNF-α and IFN-γ, and upregulate the expression of anti-inflammatory factor IL-10; by downregulating the expression levels of TLR4, COX-2 and iNOS, it can inhibit the transduction of the NF-κB pathway and reduce enteritis damage.

[0020] Furthermore, the natural oyster zinc peptide product can inhibit the decrease in expression levels of tight junction proteins Claudin-1, Occludin, and ZO-1 caused by DSS, inhibit the increase in intestinal permeability, prevent the invasion of extraintestinal substances, and protect the stability of the intestinal barrier system.

[0021] Furthermore, the natural oyster zinc peptide product can maintain the intestinal environment, improve intestinal function, and protect intestinal health by regulating the composition and diversity of intestinal flora, inhibiting the production of harmful bacteria, promoting the production of beneficial bacteria, and increasing the content of SCFAs.

[0022] Furthermore, the enteritis mentioned is ulcerative colitis.

[0023] Furthermore, the drug can be administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or locally.

[0024] The present invention also provides a medicine containing 1.25 g / kg to 5 g / kg of natural oyster zinc peptide product.

[0025] Compared with existing technologies, the effects and advantages of this invention are:

[0026] The natural oyster zinc peptide product in this invention is derived from the Pacific oyster, which is abundant and safe. Compared with commonly used drugs, the obtained natural oyster zinc peptide product is less expensive and has no toxicity or side effects.

[0027] This invention investigates the function and molecular mechanism of OZP (a natural oyster zinc peptide product) in improving ulcerative colitis. The study verifies that OZP significantly improves DSS-induced UC, not only alleviating the apparent symptoms in UC mice but also regulating them through multiple mechanisms including oxidative stress, inflammatory response, intestinal barrier function, and apoptosis. Furthermore, OZP can improve the intestinal flora structure of UC mice and increase the content of its metabolite, short-chain fatty acids. Therefore, the development of drugs using OZP for the prevention and treatment of enteritis shows great promise. Attached Figure Description

[0028] Figure 1 This is the first-order mass spectrum of OZP, a natural zinc peptide product from oysters.

[0029] Figure 2The effects of OZP on mouse body weight change and DAI score during the modeling period; (A) Mouse body weight change during the modeling period; (B) Body weight change rate on day 16; (C) DAI score; (D) DAI score on day 16;

[0030] Figure 3 The effects of OZP on the apparent and pathological parameters of the mouse colon; (A) apparent morphology of the colon; (B) colon length; (C) pathological morphology of the colon; (D) histopathological score of the colon.

[0031] Figure 4 The effects of OZP on serum inflammatory factor levels in mice; (A) TNF-α level; (B) IL-6 level; (C) IL-10 level;

[0032] Figure 5 The effects of OZP on the NF-κB inflammatory signaling pathway in colonic tissue; (A) relative expression level of TLR4 mRNA; (B) relative expression level of NF-κB mRNA; (C) relative expression level of COX-2 mRNA; (D) relative expression level of iNOS mRNA;

[0033] Figure 6 The effects of OZP on intestinal tight junction proteins in colon tissue; (A) relative expression level of Occludin mRNA; (B) relative expression level of Claudin-1 mRNA; (C) relative expression level of ZO-1 mRNA;

[0034] Figure 7 The effect of OZP on the top 30 species of abundance at the genus level in the mouse gut microbiota;

[0035] Figure 8 For the analysis of differences between groups at the genus level; (A) Analysis of differences between NC and MC groups; (B) Analysis of differences between MC and OZP groups;

[0036] Figure 9 The effect of OZP on the content of short-chain fatty acids in mice. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the scope of the examples described.

[0038] Example 1: Preparation of OZP, a natural oyster zinc peptide product

[0039] (1) Remove the shells from the oysters, wash them with deionized water, wash the oyster meat, drain it, weigh it, add 3 times the amount of deionized water (m / m), and then pour it into a tissue homogenizer to homogenize it and obtain the tissue homogenate.

[0040] (2) The tissue homogenate was ultrasonically broken up at a power of 80 W for 20 min in an ice bath to obtain tissue homogenate; the homogenate was centrifuged at 11000 r / min for 20 min and the supernatant was collected.

[0041] (3) The supernatant was concentrated using a rotary evaporator and then dried by freeze drying to obtain crude natural oyster zinc peptide product. The freeze-dried powder was stored at -20℃.

[0042] (4) The crude product of natural oyster zinc peptide was separated and purified by Sephadex G-15 gel chromatography column to obtain the natural oyster zinc peptide product. The Sephadex G-15 gel chromatography column was 100 cm long and 2.6 cm in diameter. The elution conditions were pure water and the flow rate was 0.5 mL / min.

[0043] The polypeptide molecular weight was determined to be 0.18 ± 0.06 mg / mL, and the zinc content was 377.71 ± 3.85 mg / kg. LC / MS-MS peptide sequence analysis was performed, and the mass spectrum is shown below. Figure 1 As shown, it contains four peptides with good binding ability to zinc ions: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT, and SGPSIVH.

[0044] Example 2: Establishment of a mouse UC model

[0045] Fifty-six male C57BL / 6 mice were randomly divided into seven groups after one week of acclimatization: a control group (NC), a model group (MC), a positive control group (mesalazine enteric-coated tablets, PC), high-, medium-, and low-dose OZP groups (H-OZP, M-OZP, and L-OZP), and an OZP pharmacology group (OZP), with eight mice in each group. During grouping, each mouse was observed for diarrhea or loose stools to avoid affecting subsequent experiments.

[0046] Starting from week 2, mice were treated with different doses of OZP. From day 8, mice in each experimental group were allowed free access to 3% DSS to establish the UC model, while the NC group had free access to purified water. Water bottles were replaced and sterilized every two days, and the solution inside was changed as well. Simultaneously, each group continued to receive the corresponding intervention via gavage. To minimize experimental error, gavage was performed at a fixed time each day, and all samples were freshly prepared and used immediately. Specific administration methods are shown in Table 1.

[0047] Table 1. Dosage methods for animal models

[0048]

[0049] After modeling was completed on day 17, blood was collected from the eyeballs of mice, and the serum was separated and stored at -20℃. Organs such as colon, liver, kidney, and spleen were harvested, photographed and recorded for morphology, and the organ indices were calculated after weighing. A 0.5 cm section of the distal colon was fixed for sectioning. The remaining organ tissues were weighed, wrapped in tin foil, and quickly placed in liquid nitrogen. After all sampling was completed, all samples were stored at -80℃.

[0050] Example 3

[0051] The experimental groups and gavage doses were the same as in Example 2. The weight, food intake, water intake, fecal condition and occult blood status of the mice were recorded daily, and the weight change rate and disease activity index (DAI) of the mice were calculated (Table 2).

[0052] Table 2. Disease Activity Index of Mice

[0053]

[0054] Weight change rate (%) = (Daily weight / g) / (Final weight / g) × 100%

[0055] DAI = Weight loss + Stool condition + Occult blood test

[0056] The results are as follows Figure 2 As shown, starting from day 8, mice had free access to drinking water containing 3% DSS. Changes in body weight and DAI scores were calculated. The results showed that the NC group mice experienced a slight increase in body weight while maintaining a steady level, while the MC group experienced a significant decrease in body weight starting from day 13, and began to show rectal bleeding. By day 16, the weight loss was severe, with watery stools and even rectal bleeding. After OZP treatment, the rectal bleeding and watery stools improved, weight loss decreased, and the increase in DAI scores was suppressed, indicating that OZP can improve the symptoms of ulcerative colitis.

[0057] Example 4

[0058] The experimental groups and gavage doses were the same as in Example 2. After modeling was completed on day 17, mouse colon tissue was harvested, photographed, and its length was measured. A 0.5 cm section of the distal colon was fixed for H&E section preparation. The histological damage of the colon tissue was scored according to the scoring criteria shown in Table 3.

[0059] Table 3. Colonic histopathological scores

[0060]

[0061] The results are as follows Figure 3 As shown, the apparent changes in the colon are as follows: Figure 4In mice groups A and 4B, the colons were long and uniform in thickness, with no obvious abnormalities in fecal particles and a smooth colonic surface without congestion. Compared with the NC group, the colon in the MC group showed significant congestion and unformed contents, and the colon was significantly shorter (P < 0.05). There was no significant difference in colonic condition between the OZP group and the NC group (P > 0.05), but both groups showed significant increases compared to the MC group (P < 0.05 or P < 0.01), indicating that OZP not only had no abnormal effects on mice, but also alleviated DSS-induced colonic shortening and weight loss symptoms.

[0062] Pathological changes in the colon, such as Figure 4 As shown in Figure C, the colonic mucosa of mice in the NC group was intact, with normal crypt structure and no inflammatory cell infiltration. After DSS induction, the intestinal wall of mice in the MC group thickened, the gaps in the mucus layer increased, and the mucosal tissue swelled, especially showing obvious crypt disappearance, cell vacuolation, and even ulceration. After OZP treatment, crypt disappearance decreased and the degree of mucosal tissue swelling was reduced, with the H-OZP group showing particularly significant improvement in colonic tissue.

[0063] The results of colonic pathology scoring are as follows: Figure 4 As shown in Figure D, compared with the NC group, the MC group showed a highly significant increase in scores (P < 0.01), indicating that the MC group mice had greater colonic damage and a more severe inflammatory response. OZP treatment significantly reduced tissue damage (P < 0.01), with H-OZP showing the most significant improvement in colonic tissue.

[0064] Example 5

[0065] The experimental groups and gavage doses were the same as in Example 2. After modeling was completed on day 17, blood was collected from the eyes of the mice, and the serum was separated and stored at -20°C. The levels of inflammatory cytokines in the blood were detected according to the TNF-α, IL-6, and IL-10 kit instructions.

[0066] The results are as follows Figure 4 As shown, OZP can reduce the body's inflammation level by inhibiting the secretion of pro-inflammatory cytokines IL-6 and TNF-α and promoting the secretion of anti-inflammatory factor IL-10, thereby alleviating the damage caused by UC to the body.

[0067] Example 6

[0068] The experimental groups and gavage doses were the same as in Example 2. After the modeling was completed on day 17, the colon was taken, the morphology was photographed and recorded, the distal colon was fixed and sectioned, the remaining colon tissue was weighed, wrapped in tin foil and quickly placed in liquid nitrogen, and after the sampling was completed, it was uniformly stored at -80℃.

[0069] The remaining colon tissue was used for subsequent experiments. Total RNA was extracted according to the Transzol up instructions, and RNA integrity, concentration, and purity were determined. The reaction system was constructed according to Table 4, and reverse transcription of RNA was performed.

[0070] Table 4 PCR reaction system

[0071]

[0072] PCR reaction conditions: 42 ℃ for 30 min, then 85 ℃ for 5 s. The resulting cDNA was stored at -20 ℃ for later use.

[0073] Following the instructions for TransStart® Top Green qPCR SuperMix (+Dye II), add the reagents as shown in Table 5 to 100 μL eight-tube bundles, mix well, and centrifuge.

[0074] Table 5 qRT-PCR reaction system

[0075]

[0076] qPCR was performed using a Roche fluorescence spectrometer, with the following program settings:

[0077]

[0078] Using β-actin as an internal reference, Ct(2) -△△CT The expression levels of the target genes were calculated. The mRNA expression levels of TLR4, NF-κB, COX-2 and iNOS, which are related to the NF-κB signaling pathway, were detected in mouse colon tissue. The primer sequences are shown in Table 6.

[0079] Table 6 qRT-PCR primer sequences

[0080]

[0081] The results are as follows Figure 5 As shown, compared with the NC group, the mRNA expression levels of NF-κB pathway-related genes in the MC group were significantly increased (P < 0.01). After OZP treatment, the mRNA expression levels of related genes all showed a decreasing trend, with the L-OZP group showing the best improvement (P < 0.01).

[0082] Example 7

[0083] The same method as in Example 6 was used to extract RNA and perform quantitative real-time PCR analysis to detect the mRNA expression levels of the intestinal tight junction proteins Occludin, Claudin-1, and ZO-1 in mouse colon tissue. The primer sequences are shown in Table 7.

[0084] Table 7 qRT-PCR primer sequences

[0085]

[0086] The results are as follows Figure 6 As shown, compared with the MC group, the mRNA level of TJs in the MC group was significantly reduced (P < 0.01). After OZP treatment, the expression level of TJs was significantly upregulated (P < 0.05 or P < 0.01), and the OZP group had a highly significant upregulating effect on the expression of transmembrane proteins Claudin-1 and Occludin mRNA (P < 0.01).

[0087] Example 8

[0088] The experimental groups and gavage doses were the same as in Example 2. At day 16, feces from each mouse were collected and stored in sterile, enzyme-inactivated cryovials for 16S rDNA amplicon sequencing, detecting the V3-V4 region. The sequencing platform was an Illumina Miseq PE300. Reads were filtered by shearing, OTUs (Operational Taxonomic Units) were clustered, and species annotation and abundance analysis were performed. The Kruskal-Wallis rank-sum test was used to analyze species differences among multiple groups.

[0089] The short-chain fatty acid content of each group of samples was determined by GC-MS. The analysis was performed using an HPFFAP capillary column (30 m × 0.25 mm × 0.25 μm), and other analytical conditions are shown in Table 8. Finally, the ion fragments were automatically identified and integrated using Masshunter quantitative software.

[0090] Table 8 GC-MS Analysis Conditions

[0091]

[0092] genus-level species composition results as follows Figure 7 As shown, OZP can regulate the gut microbiota structure of UC mice by promoting Lachnospiraceae_NK4A136_group , Allobaculum The production of beneficial bacteria inhibits Helicobacter , Escherichia-Shigella The growth of harmful bacteria helps maintain intestinal homeostasis and alleviate UC symptoms.

[0093] Between-group difference analysis, such as Figure 8 As shown, at the genus level, compared with the NC group, the MC group mice... Dubosiella , norank_f_norank_o_Clostridia_UCG-014 , Erysipelatoclostridium , Romboutsia , Turicibacter , unclassified_f_Peptostreptococcaceae , norank_f__norank_o__RF39 The content of fungi increased significantly. Helicobacter , Rikenella , Candidatus_Arthromitus The bacterial count decreased significantly; compared with the OZP group, the MC group mice showed a significant decrease. Coriobacteriaceae_UCG-002 , Butyricicoccus , unclassified_c__ Clostridia The number of fungal species increased significantly. Rikenellaceae_RC9_gut_group and norank_f__Mitochondria The number of bacterial species decreased significantly.

[0094] Short-chain fatty acid determination results are as follows Figure 9 As shown, after DSS treatment, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, isohexanoic acid, and total SCFA in the MC group were significantly reduced. After OZP treatment, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and total SCFA were significantly increased (P < 0.01).

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The application of a natural oyster zinc peptide product in the preparation of a drug for the prevention and treatment of enteritis, characterized in that, The enteritis is ulcerative colitis, and the preparation method of the natural oyster zinc peptide product is as follows: (1) Remove the shells from the oysters, clean the oyster meat, add 2 to 4 times the volume of deionized water and crush to obtain the crushed tissue fluid; (2) The tissue homogenate from step (1) is subjected to ultrasonic disruption, and the supernatant is collected by centrifugation. The ultrasonic disruption power is 80 W and the time is 20 min. (3) The supernatant from step (2) was rotary evaporated and freeze-dried to obtain crude natural oyster zinc peptide product; (4) The crude natural oyster zinc peptide product from step (3) is separated and purified to obtain the natural oyster zinc peptide product. The separation and purification are performed using a Sephadex G-15 gel chromatography column, which is 100 cm long and 2.6 cm in diameter. The elution conditions are pure water and the flow rate is 0.5 mL / min.

2. The application according to claim 1, characterized in that, The drug can be administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, or intradermally.

3. The application according to claim 1, characterized in that, The drug can be administered via local administration.

4. The application according to claim 1, characterized in that, The natural oyster zinc peptide product contains four peptide segments; the amino acid sequences of the peptide segments are: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT, and SGPSIVH.

5. A drug, characterized in that, The drug contains 1.25 g / kg to 5 g / kg of the natural oyster zinc peptide product as described in claim 1.

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