CN116236558A - Application of natural oyster zinc peptide product in preparation of medicine or health-care product for preventing and treating enteritis - Google Patents
Application of natural oyster zinc peptide product in preparation of medicine or health-care product for preventing and treating enteritis Download PDFInfo
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- CN116236558A CN116236558A CN202211098709.0A CN202211098709A CN116236558A CN 116236558 A CN116236558 A CN 116236558A CN 202211098709 A CN202211098709 A CN 202211098709A CN 116236558 A CN116236558 A CN 116236558A
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses an application of a natural oyster zinc peptide product in preparing a medicine or health care product for preventing and treating enteritis. According to the invention, animal experiments prove that the natural oyster zinc peptide product has the functions of preventing and treating enteritis, and can improve the apparent symptoms of ulcerative colitis of mice caused by DSS by regulating the level of oxidative stress, relieving inflammatory reaction, maintaining intestinal barrier and reducing apoptosis, and can also regulate the intestinal flora and metabolic products of enteritis mice, so that intestinal inflammation is relieved. The natural oyster zinc peptide product used by the invention has good safety, lower cost and no side effect compared with the common anti-inflammatory and immunity-enhancing medicaments, and has remarkable effect of relieving enteritis. Therefore, the natural oyster zinc peptide product has good prospect in the aspect of developing medicines or health care products for preventing and treating enteritis.
Description
Technical Field
The invention belongs to the field of food health care, and in particular relates to application of a natural oyster zinc peptide product in preparation of a medicine or health care product for preventing and treating enteritis.
Background
In recent years, inflammatory Bowel Disease (IBD) has become one of the most serious chronic diseases that endanger human health. Symptoms of IBD are often manifested by inflammation of the colon or rectum, ulcers, diarrhea, hematochezia, and weight loss, and a long course of disease. Among them, recurrence of Ulcerative Colitis (UC) is often accompanied by complications, severely affecting the quality of life of the patient. Research data show that the incidence of UC tends to rise year by year, especially in developed countries, but drugs for treating UC are often expensive and have many side effects, such as acne, abdominal pain, weight gain, diabetes, etc., so it is imperative to find functional components for treating or relieving UC from food-borne active substances.
In order to explore the pathogenesis of IBD that may exist, new targets for disease treatment were sought, and different animal models were applied in experiments. Among these, DSS-induced UC animal models are most commonly used. Animals can develop UC-like symptoms by adding DSS to drinking water, which is one of the ideal animal models for studying the efficacy of IBD treatment. DSS has toxic effect on colon epithelial cells, can destroy intestinal barrier, increase intestinal permeability, promote inflammatory cytokines and toxins to enter intestinal tract through intestinal wall, activate related inflammatory signal pathway, and cause inflammatory injury, so that experimental animals show obvious symptoms such as weight reduction, diarrhea, hematochezia and hypoactivity. The method has the advantages of convenient operation, strong repeatability and most wide application in enteritis models.
More and more research reports indicate that the beneficial effects of a large number of food ingredients, nutritional ingredients such as proteins, active peptides and the like on UC far exceed the traditional nutritional value. Proteins and the like can be used as carbon sources and nitrogen sources of intestinal microorganisms for growth, and the microorganisms can generate SCFAs after metabolism, thereby participating in regulation of inflammatory reactions. The functional research of oyster bioactive peptide is increasingly abundant, the biological utilization rate of zinc ions can be improved, and zinc element has certain anti-inflammatory activity. The natural oyster zinc peptide product prepared by the invention is prepared from crassostrea gigas, has rich raw materials, good safety and no side effect, and therefore, has great potential in the development of medicines or health care products for preventing and treating enteritis.
Disclosure of Invention
The invention provides an application of a natural oyster zinc peptide product in preparing a medicine or health care product for preventing and treating enteritis. The natural oyster zinc peptide product has rich and easily obtained raw materials, good safety and obvious enteritis improvement effect, and can regulate intestinal flora and metabolites of enteritis mice.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides an application of a natural oyster zinc peptide product in preparing a medicine or health care product for preventing and treating enteritis.
Furthermore, the natural oyster zinc peptide product contains 4 peptide segments with better binding ability with zinc ions; the amino acid sequences of the peptide fragments are respectively as follows: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH.
Further, the preparation method of the natural oyster zinc peptide product comprises the following steps:
(1) Removing shells of oysters, cleaning oyster meat, and mashing with deionized water with the volume of 2-4 times to obtain mashed tissue fluid;
(2) Performing ultrasonic crushing on the tissue triturating liquid obtained in the step (1), and centrifugally collecting supernatant;
(3) Spin-steaming the supernatant in the step (2), and freeze-drying to obtain a crude product of the natural oyster zinc peptide;
(4) And (3) separating and purifying the crude product of the natural oyster zinc peptide in the step (3) to obtain the natural oyster zinc peptide product.
Further, the ultrasonic crushing power in the step (2) is 70W-100W, and the time is 15min-30min.
Further, the effective dose of the natural oyster zinc peptide product is 1.25g/kg/bw-5g/kg/bw.
Furthermore, the natural oyster zinc peptide product can obviously reduce the weight loss of mice caused by DSS, improve the abnormal condition of fecal state and inhibit the increase of DAI scores of the mice.
Furthermore, the natural oyster zinc peptide product can relieve colon appearance and internal tissue lesions caused by DSS induction, maintain the integrity of colon tissues of mice and play a positive role in stabilizing the intestinal tract environment.
Furthermore, the natural oyster zinc peptide product can achieve the effect of preventing and treating enteritis by down regulating the expression of pro-inflammatory factors, mediating the activation of NF- κB channels, regulating intestinal flora structure and increasing the content of SCFAs.
Further, the natural oyster zinc peptide product can down regulate the expression of pro-inflammatory factors IL-6, TNF-alpha and IFN-gamma, up regulate the expression of anti-inflammatory factors IL-10; by down regulating expression level of TLR4, COX-2 and iNOS, transduction of NF- κB pathway is inhibited, and enteritis injury is reduced.
Furthermore, the natural oyster zinc peptide product can inhibit the reduction of the expression level of Claudin-1, occludin and ZO-1 which are compact proteins caused by DSS, inhibit the increase of intestinal permeability, prevent the invasion of substances outside the intestines and protect the stability of an intestinal barrier system.
Furthermore, the natural oyster zinc peptide product can maintain the environment in the intestinal tract, improve the intestinal tract function and protect the health of the intestinal tract by regulating the composition and diversity of intestinal flora, inhibiting the generation of harmful bacteria, promoting the generation of beneficial bacteria and increasing the content of SCFAs.
Further, the enteritis is ulcerative colitis.
Further, the drug or health product can be administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically.
The invention also provides a medicine or health care product, which contains 1.25g/kg-5g/kg of natural oyster zinc peptide product.
Compared with the prior art, the invention has the following effects and advantages:
the natural oyster zinc peptide product is derived from crassostrea gigas, has rich raw materials and good safety, and has lower cost, no toxicity or side effect compared with common medicines.
According to the invention, through research on the function and molecular mechanism of the natural oyster zinc peptide product OZP for improving ulcerative colitis, the effect of the natural oyster zinc peptide product OZP for improving DSS-induced UC is proved to be obvious, the apparent symptoms of UC mice are relieved, and the UC mice are regulated through a plurality of aspects such as oxidative stress, inflammatory reaction, intestinal barrier, apoptosis and the like, in addition, the intestinal flora structure of the UC mice can be improved by OZP, and the content of metabolic products short-chain fatty acids of the UC mice is increased. Therefore, OZP has good prospect in the development of medicines or health care products for preventing and treating enteritis.
Drawings
FIG. 1 is a first-order mass spectrum of a natural oyster zinc peptide product OZP;
FIG. 2 is a graph showing the effect of OZP on mouse weight change and DAI score during molding; (a) weight change in mice during molding; (B) day 16 body weight change rate; (C) DAI scoring; (D) day 16 DAI score;
FIG. 3 shows OZP colon appearance index and pathological impact on mice; (a) colon appearance; (B) colon length; (C) colon pathology; (D) colon histopathological scoring;
FIG. 4 is a graph of the effect of OZP on serum inflammatory factor levels in mice; (a) TNF- α content; (B) IL-6 content; (C) IL-10 content;
FIG. 5 is a graph of OZP effect on NF- κB inflammatory signaling pathways in colon tissue; (a) relative amount of TLR4 mRNA expression; (B) relative expression amount of NF- κB mRNA; (C) relative expression level of COX-2 mRNA; (D) the relative expression amount of iNOS mRNA;
FIG. 6 is a graph of the effect of OZP on intestinal claudin in colon tissue; (A) the relative expression amount of Occludin mRNA; (B) relative expression amount of Claudin-1 mRNA; (C) ZO-1mRNA relative expression amount;
FIG. 7 is a graph of the effect of OZP on the pre-30 species of mice gut flora level abundance;
FIG. 8 is an analysis of inter-group differences at the genus level; (a) analysis of NC and MC group differences; (B) differential analysis of MC and OZP groups;
figure 9 is the effect of OZP on short chain fatty acid content in mice.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description, but the scope of the invention is not limited to the scope expressed by examples.
Example 1: preparation of natural oyster zinc peptide product OZP
(1) Removing shells of crassostrea gigas, cleaning with deionized water, cleaning oyster meat, draining, weighing, adding 3 times of deionized water (m/m), and then pouring into a tissue masher for mashing to obtain tissue mashing liquid;
(2) Carrying out ultrasonic crushing on the tissue smashing liquid with the power of 80W and the time of 20min, wherein the crushing process is carried out in an ice bath to obtain tissue smashing liquid; centrifuging the crushed solution at 11000r/min for 20min, and collecting supernatant;
(3) Concentrating the supernatant by using a rotary evaporator, drying the supernatant by using a freeze-drying method to obtain crude natural oyster zinc peptide products, and preserving freeze-dried powder at-20 ℃;
(4) And (3) separating and purifying the crude product of the natural oyster zinc peptide by using a Sephadex G-15 gel chromatographic column to obtain the natural oyster zinc peptide, wherein the Sephadex G-15 gel chromatographic column has the length of 100cm and the diameter of 2.6cm, the eluent condition is pure water, and the flow rate is 0.5mL/min.
The polypeptide molecular weight of the natural oyster zinc peptide product is measured, the polypeptide content is 0.18+/-0.06 mg/mL, the zinc content is 377.71 +/-3.85 mg/kg, and the mass spectrum is shown as figure 1 through LC/MS-MS peptide fragment sequence analysis, and the natural oyster zinc peptide product contains 4 peptide fragments with better binding capacity with zinc ions: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH.
Example 2: establishment of mouse UC model
After one week of adaptive feeding, 56C 57BL/6 male mice were randomly divided into 7 groups: normal (control, NC), model (MC), positive (positive, PC) and high, medium and low dose OZP (H-OZP, M-OZP and L-OZP) and OZP pharmacological groups (OZP), 8 each. During grouping, whether diarrhea and loose stool exist in each mouse is observed, and the influence on subsequent experiments is avoided.
Different doses of OZP intervention were performed starting from week 2. Starting from day 8, the mice of each experimental group are allowed to drink 3% DSS freely to establish a UC model, the NC group is allowed to drink purified water freely, the drinking bottle is replaced and disinfected every 2 days, and the solution in the bottle is replaced; at the same time, each group continues to perfuse the stomach with the corresponding intervention. To reduce experimental errors, the lavage was performed at fixed times per day, with all samples ready for use. The specific modes of administration are shown in Table 1.
Table 1 animal model administration mode
After the molding was completed on day 17, the mice were subjected to eyeball blood collection, and serum was isolated and stored at-20 ℃. Taking colon, liver, kidney, spleen and other organs, photographing and recording forms, weighing the organs, and calculating an organ index; fixing the distal colon by 0.5cm, and making slices; weighing the rest organ tissues, wrapping with tinfoil, rapidly adding liquid nitrogen, and uniformly storing at-80deg.C after the materials are obtained.
Example 3
The experimental group and the gastric lavage dose were the same as in example 2, the weight, food intake, water intake, fecal status and occult blood condition of the mice were recorded daily, and the weight change rate and disease activity index (Disease activity index, DAI) of the mice were calculated (table 2).
TABLE 2 Activity of mice disease index table
Weight change rate (%) = (weight on day/g)/(final weight/g) ×100%
DAI = weight loss + fecal status + occult blood condition
As a result, as shown in fig. 2, from day 8, mice were free to drink 3% dss-containing drinking water, and the weight change and DAI score change were calculated to find that the weight of NC group mice slightly increased while keeping gentle, whereas the weight of MC group significantly decreased from day 13 and began to show a hematochezia condition, to a serious decrease in weight from day 16, watery stool, and even rectal bleeding. After OZP treatment, the conditions of hematochezia and watery stool are improved, the weight loss is reduced, and the DAI score is inhibited from rising, so that OZP can improve the symptoms of ulcerative enteritis.
Example 4
Experiment groups and gastric lavage doses are the same as in example 2, after the molding is finished on the 17 th day, colon tissues of the mice are taken, morphology is recorded by photographing, and the length of the colon tissues is measured; the distal colon was fixed 0.5cm and used to make H & E sections. The histological damage to the colon was scored, and the scoring criteria are shown in table 3.
Table 3 colon histopathological scoring
As shown in FIG. 3, the apparent changes of the colon are shown in FIG. 3A and FIG. 3B, the colon of the NC group mice is slender and uniform in thickness, fecal particles in the colon are obviously free from abnormality, and the surface of the colon is smooth and free from congestion. The colonic hyperemia was evident and the contents were unshaped, and the length of the colon was significantly shortened (P < 0.05) compared to NC group. The colon condition of OZP mice is not obviously different from that of NC mice (P is more than 0.05), and compared with MC mice, the colon condition of the mice is obviously increased (P is less than 0.05 or P is less than 0.01), which indicates that OZP has no abnormal influence on the mice, and OZP can relieve DSS-induced colon shortening and weight reduction symptoms.
The colon pathological change is shown in fig. 3C, the colon mucosa of the mice in the NC group is complete, the crypt structure is normal, inflammatory cells are not infiltrated, after DSS induction, the intestinal wall of the mice in the MC group is thickened, the gap of the mucus layer is enlarged, the mucous membrane tissue is swollen, and especially obvious crypt disappearance, cell cavitation and even ulceration are caused. After OZP treatment, the crypt disappeared and the swelling degree of the mucous membrane tissue was reduced, and the improvement effect of the colon tissue of the H-OZP group was particularly obvious.
The results of scoring colon pathology are shown in fig. 3D, and the score of the MC group is extremely significantly increased (P < 0.01) compared with the NC group, indicating that the colon injury of the MC group mice is larger and the inflammatory response is aggravated. OZP treatment significantly reduced tissue damage (P < 0.01), with H-OZP having the most pronounced effect on improving colon tissue.
Example 5
Experiment groups and gastric lavage doses were the same as in example 2, and after the end of the molding on day 17, mice were subjected to eyeball blood collection and serum separation for storage at-20 ℃. According to the instruction book of the TNF-alpha, IL-6 and IL-10 kit, the inflammatory cytokine content in blood is detected.
As shown in FIG. 4, OZP can reduce the inflammatory level of the organism by inhibiting the secretion of pro-inflammatory cytokines IL-6 and TNF-alpha, promote the secretion of anti-inflammatory factors IL-10, and thus relieve the damage of UC to the organism.
Example 6
Experiment grouping and stomach filling dosage are the same as in example 2, after the molding is finished on the 17 th day, taking the colon, photographing and recording the shape, taking the far-end colon for fixing, and making slices; weighing the rest colon tissue, wrapping with tinfoil, rapidly adding liquid nitrogen, and uniformly storing at-80deg.C after the materials are obtained.
Subsequent experiments were performed using the remaining colon tissue, total RNA was extracted according to the Transzol up instructions, and RNA integrity, concentration and purity determinations were performed. A reaction system was constructed in accordance with Table 4, and reverse transcription of RNA was performed.
TABLE 4 PCR reaction System
PCR reaction conditions: 42 ℃,30min, then 85 ℃,5s, and the obtained cDNA is stored at-20 ℃ for standby.
Reference to
Top Green qPCR SuperMix (+dye II) instructions, the reagents were added to 100. Mu.L of eight-row tubes according to Table 5, mixed well and centrifuged.
TABLE 5 qRT-PCR reaction System
qPCR was performed using a rogowski fluorometer with the following procedure:
beta-actin is used as an internal reference, ct (2 -△△CT ) And calculating the expression quantity of the target gene. mRNA expression levels of related genes TLR4, NF- κ B, COX-2 and iNOS of the colon tissue NF- κB signaling pathway of the mice are detected, and primer sequences are shown in Table 6.
TABLE 6 qRT-PCR primer sequences
As a result, as shown in FIG. 5, the mRNA expression level of the NF- κB pathway-related gene in the MC group was extremely significantly increased (P < 0.01) as compared with the NC group. After OZP treatment, the mRNA expression levels of the related genes all showed a decreasing trend, and the improvement effect of the L-OZP group was the best (P < 0.01).
Example 7
The mRNA expression levels of mouse colon tissue intestinal tract zonulin Occludin, claudin-1 and ZO-1 were measured by the same method as in example 6 and by fluorescence quantitative PCR analysis, and the primer sequences thereof are shown in Table 7.
TABLE 7 qRT-PCR primer sequences
As a result, as shown in FIG. 6, the mRNA level of the MC group TJs was significantly reduced (P < 0.01) as compared with the MC group. After OZP treatment, TJs expression levels were significantly up-regulated (P < 0.05 or P < 0.01), with OZP group exerting a very significant up-regulation effect on transmembrane protein Claudin-1, occludin mRNA expression (P < 0.01).
Example 8
Experiment group and stomach filling dose were the same as in example 2, and feces from each mouse were collected at 16 days in sterilized and enzyme-inactivated cryopreservation tubes for 16SrDNA amplicon sequencing, V3-V4 regions were detected, and the sequencing platform was Illumina Miseq PE. Species difference analysis between groups was performed by shear filtration on Reads, OTUs (Operational Taxonomic Units) clustering, and species annotation and abundance analysis using Kruskal-Wallis rank sum test.
The short chain fatty acid content of each group of samples was detected by GC-MS. The analysis uses HPFFAP capillary columns (30 m 0.25mm 0.25 μm) and other analysis conditions are shown in Table 8. Finally, the ion fragments were automatically identified and integrated by Masshunter quantification software.
TABLE 8 GC-MS analysis conditions
The results of the composition of the genus level species are shown in fig. 7, OZP can regulate the intestinal flora structure of UC mice, inhibit the production of harmful bacteria such as Helicobacter, escherichia-Shigella and the like by promoting the production of beneficial bacteria such as Lachnospiraceae_NK4A136_ group, allobaculum and the like, maintain intestinal homeostasis and relieve UC symptoms.
As shown in fig. 8, the inter-group difference analysis showed that, at the genus level, the MC group mice had significantly increased levels of the Dubosiella, norank _f_norank_o_clostridium_ucg-014, erysipelatoclostridium, romboutsia, turicibacter, unclassified _f_ Peptostreptococcaceae, norank _f __ norank_o __ RF39 bacteria and significantly decreased levels of the Helicobacter, rikenella, candidatus _archomitus bacteria compared to the NC group; compared to group OZP, the MC group mice had significantly increased strains of Corynebacterium_UCG-002, butyricicoccus, unclassified _c __ Clostridia, and significantly decreased strains of Rikenella ceae_Rc9_gun_group and nonrank_f __ Mitochondronia.
The short chain fatty acid assay results are shown in figure 9, and the acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, isocaproic acid and total SCFA content of the MC group were significantly reduced after DSS treatment. 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).
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. Application of natural oyster zinc peptide product in preparing medicine or health product for preventing and treating enteritis.
2. The use according to claim 1, wherein the natural oyster zinc peptide product contains 4 peptide fragments; the amino acid sequences of the peptide fragments are respectively as follows: EKSGPGPHCPRC, TVGPITGGGSHK, VIDTNKDRT and SGPSIVH.
3. The use according to claim 1, wherein the preparation method of the natural oyster zinc peptide product is as follows:
(1) Removing shells of oysters, cleaning oyster meat, and mashing with deionized water with the volume of 2-4 times to obtain mashed tissue fluid;
(2) Performing ultrasonic crushing on the tissue triturating liquid obtained in the step (1), and centrifugally collecting supernatant;
(3) Spin-steaming the supernatant in the step (2), and freeze-drying to obtain a crude product of the natural oyster zinc peptide;
(4) And (3) separating and purifying the crude product of the natural oyster zinc peptide in the step (3) to obtain the natural oyster zinc peptide product.
4. The use according to claim 3, wherein the power of the ultrasonic disruption in step (2) is 70W-100W for 15min-30min.
5. The use according to claim 1, wherein the effective dose of the natural oyster zinc peptide product is 1.25g/kg/bw-5g/kg/bw.
6. The use according to claim 1, wherein the natural oyster zinc peptide product is capable of significantly reducing weight loss in mice caused by DSS, ameliorating abnormal fecal conditions, and inhibiting elevation of DAI score in mice.
7. The use according to claim 1, wherein the natural oyster zinc peptide product is capable of achieving the effect of preventing and treating enteritis by down-regulating the expression of pro-inflammatory factors, mediating the activation of NF- κb pathway, regulating intestinal flora structure and increasing SCFAs content.
8. The use according to claim 1, wherein the enteritis is ulcerative colitis.
9. The use according to claim 1, wherein the medicament or health product is capable of oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration or topical administration.
10. A medicine or health care product is characterized in that the medicine or health care product contains 1.25g/kg-5g/kg of natural oyster zinc peptide product.
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