A sea cucumber intestinal polypeptide and its application in preparing medicine for gastric diseases

Abstract

The present invention discloses a sea cucumber intestinal polypeptide and its application in the preparation of gastric disease drugs, and relates to the field of biomedicine technology. A sea cucumber intestinal polypeptide, whose amino acid sequence includes one or more of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The present invention uses compound papain and neutral protease to hydrolyze sea cucumber intestine, takes the degree of hydrolysis as the evaluation standard, and determines the optimal hydrolysis conditions of sea cucumber intestine through single factor and response surface experiments, and screens out three sea cucumber intestinal polypeptides with the best in vitro antioxidant activity from the sea cucumber intestinal polypeptides prepared by enzymatic hydrolysis. The screened sea cucumber intestinal polypeptides have good application prospects in the treatment and prevention of gastric diseases caused by damaged gastric mucosa.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a sea cucumber intestinal polypeptide and application thereof in the preparation of a medicine for treating gastric diseases. Background Art

[0002] With the accelerated pace of life and the increase of pressure, drinking has become a daily habit of people, but excessive drinking may cause damage to the gastric mucosa and cause alcohol-related gastritis. For gastric diseases caused by gastric mucosal damage, chemical drugs such as omeprazole are generally used for treatment. However, long-term use of proton pump inhibitors such as omeprazole for gastric disease treatment, although it can effectively relieve gastric symptoms, may also bring some adverse effects.

[0003] Studies have found that substances such as peptides in sea cucumber intestines have multiple biological activities such as anti-oxidation, hypoglycemic, immune enhancement, and bone growth promotion. In addition, bioactive peptides have the advantages of mild performance, wide sources, easy solubility, easy absorption, and extremely low toxicity. Compared with other bioactive substances, low molecular weight peptides have high activity, stability, and specificity. As a natural active ingredient, food-derived bioactive peptides have received widespread attention.

[0004] In this regard, the inventors believe that how to screen out a sea cucumber intestinal polypeptide from sea cucumber intestines for treating and preventing gastric diseases caused by damaged gastric mucosa, and that this sea cucumber intestinal polypeptide must have good biological activity, is a technical problem that the technicians in this field urgently need to solve.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] In view of the above technical problems, an embodiment of the present invention provides a sea cucumber intestinal polypeptide and its application in the preparation of gastric disease drugs to solve the problems raised in the above background technology.

[0007] A sea cucumber intestinal polypeptide, whose amino acid sequence comprises one or more of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; wherein SEQ ID NO.1 is WRPPPP; SEQ ID NO.2 is SHFFR; and SEQ ID NO.3 is LPPR.

[0008] Preferably, the sea cucumber intestine polypeptide is prepared from sea cucumber intestine by enzymolysis of the sea cucumber intestine using papain and neutral protease in a mass ratio of 1:1.

[0009] Preferably, the enzymatic hydrolysis conditions are: temperature 54°C, enzyme addition amount 4454U / g, and time 3h.

[0010] A use of the sea cucumber intestinal polypeptide as described above in the preparation of a drug for treating and / or preventing gastric diseases.

[0011] Preferably, the gastric disease is a gastric disease caused by damaged gastric mucosa.

[0012] A pharmaceutical composition for treating and / or preventing gastric diseases, comprising the sea cucumber intestinal polypeptide or a pharmaceutically acceptable salt of the sea cucumber intestinal polypeptide as described above.

[0013] Preferably, the pharmaceutical composition further comprises one or more therapeutic agents; the therapeutic agents include drugs having a protective effect on the gastric mucosa.

[0014] Preferably, the composition also includes at least one of a pharmaceutically acceptable carrier, excipient, adjuvant and vehicle.

[0015] Preferably, the dosage form of the pharmaceutical composition is an oral preparation, a mucosal administration preparation or an injection.

[0016] Preferably, the pharmaceutical composition is used for protecting and repairing gastric mucosal damage.

[0017] A sea cucumber intestinal polypeptide provided by an embodiment of the present invention and its use in the preparation of gastric disease drugs have the following beneficial effects: the present invention selects a compound papain and a neutral protease to hydrolyze the sea cucumber intestine, takes the hydrolysis degree as the evaluation standard, and determines the optimal hydrolysis conditions of the sea cucumber intestine through single factor and response surface experiments, and screens out three sea cucumber intestinal polypeptides with the best in vitro antioxidant activity from the sea cucumber intestinal polypeptides prepared by enzymatic hydrolysis. The screened sea cucumber intestinal polypeptides have good application prospects in the treatment and prevention of gastric diseases caused by damaged gastric mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the effect of a single factor on the degree of hydrolysis in the present invention;

[0019] in, Figure 1 In a, time is a single factor variable; Figure 1 In b, the amount of enzyme added is a single factor variable; Figure 1 In c, temperature is a single factor variable;

[0020] Figure 2 It is the response surface analysis result of the present invention;

[0021] in, Figure 2 In a, the effect of the interaction between temperature and time on the degree of hydrolysis; Figure 2 In b, the effect of the interaction between temperature and enzyme addition on the degree of hydrolysis; Figure 2In c, the effect of the interaction between time and enzyme addition amount on the degree of hydrolysis;

[0022] Figure 3 The results are for the antioxidant activity of sea cucumber intestinal peptides with different molecular weights;

[0023] in, Figure 3 a, the results of DPPH free radical scavenging rate of sea cucumber intestinal polypeptides with different molecular weights; Figure 3 b shows the superoxide anion free radical inhibition rate results of sea cucumber intestinal polypeptides with different molecular weights; Figure 3 c is the result of scavenging rate of hydroxyl radicals of sea cucumber intestinal polypeptides with different molecular weights;

[0024] Figure 4 This is the result diagram of establishing the ethanol-induced GES-1 cell injury model;

[0025] Figure 5 This is the result diagram of the effect of sea cucumber intestinal peptide on the activity of GES-1 cells;

[0026] Figure 6 This is the result diagram of the effect of sea cucumber intestinal peptide on the proliferation of GES-1 cells damaged by ethanol;

[0027] Figure 7 This is the result diagram of the effect of sea cucumber intestinal peptide on ROS in ethanol-damaged GES-1 cells;

[0028] Figure 8 This is the result diagram of the effect of sea cucumber intestinal peptide on the activity of SOD enzyme in damaged GES-1 cells;

[0029] Fig. 9 This is the result diagram of the effect of sea cucumber intestinal peptide on the MDA content in damaged GES-1 cells;

[0030] Fig.10 a is the result of separation and purification of sea cucumber intestinal peptide;

[0031] Fig.10 b, 10c, and 10d are graphs showing the results of DPPH radical scavenging, superoxide anion radical inhibition, and hydroxyl radical inhibition by different components, respectively;

[0032] Fig.11 is the secondary structure mass spectrum of WRPPPP;

[0033] Fig.12 is the secondary structure mass spectrum of SHFFR;

[0034] Fig.13 This is the mass spectrum of the secondary structure of LPPR. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0036] In view of the above technical problems, an embodiment of the present invention provides a sea cucumber intestinal polypeptide and its application in the preparation of gastric disease drugs to solve the problems raised in the above background technology.

[0037] 1 Materials and methods

[0038] 1.1 Materials and reagents

[0039] Sea cucumber intestines were collected in Linghai City, Jinzhou, and stored at -20°C for future use. Papain (800,000 U / g) and neutral protease (50,000 U / g) were from Beijing Solebaugh Technology Co., Ltd.; all kits were from Nanjing Jiancheng Bioengineering Institute.

[0040] 1.2 Test methods

[0041] 1.2.1 Preparation of sea cucumber intestinal peptide

[0042] 1.2.1.1 Determination of degree of hydrolysis

[0043] Add 6 times the mass of water homogenate, add papain and neutral protease in a 1:1 ratio, and perform enzymolysis in a constant temperature water bath oscillator. After the enzymolysis is completed, inactivate the enzyme at 95°C for 15 minutes, centrifuge at 10,000 rpm for 20 minutes, collect the supernatant, and calculate the degree of hydrolysis of the sea cucumber intestine enzymolysate according to formula (1):

[0044] Degree of hydrolysis (DH / %) = amino acid nitrogen content / total nitrogen content × 100 (1)

[0045] Wherein: the amino acid nitrogen content is determined according to the acidometer method (GB 5009.235-2016); the total nitrogen content is determined according to the Kjeldahl nitrogen method (GB 5009.5-2016).

[0046] 1.2.1.2 Single Factor Experiment

[0047] Taking the enzymatic hydrolysis temperature of 60℃, the enzyme dosage of 3000U / g and the time of 3.5h as the basic conditions and the hydrolysis degree as the evaluation basis, the three factors with the greatest influence on the hydrolysis degree of sea cucumber intestine, namely enzymatic hydrolysis temperature (40, 50, 60, 70, 80℃), enzyme dosage (1000, 2000, 3000, 4000, 5000U / g) and time (2.5, 3.0, 3.5, 4.0, 4.5h), were optimized respectively.

[0048] 1.2.1.3 Response surface experiment

[0049] On the basis of single factor experiments, enzymatic hydrolysis temperature, enzyme dosage and enzymatic hydrolysis time were the key variables affecting the degree of hydrolysis. The Box-Behnken method was used to design a response surface experiment with three factors and three levels to optimize the enzymatic hydrolysis conditions.

[0050] 1.2.1.4 Ultrafiltration separation

[0051] The enzymatic hydrolysate was filtered with 0.45μm and 0.22μm microporous membranes, and then added to a reverse osmosis separation test machine for ultrafiltration classification. The pressure was controlled at 0.4MPa, and the filtrate and the retentate were collected to prepare three different molecular weight polypeptides of >3KDa, 1-3KDa, and <1KDa (P1, P2, and P3). Vacuum freeze drying was performed to obtain different molecular weight polypeptide powders of sea cucumber intestines.

[0052] 1.2.1.5 Determination of the antioxidant activity of sea cucumber intestinal polypeptides in vitro

[0053] Different concentrations of P1, P2, and P3 solutions (5, 10, 15, 20, and 25 mg / mL) were prepared, and the DPPH free radical scavenging ability, inhibition and generation of superoxide anion free radicals, and hydroxyl free radicals were measured and calculated according to the kit instructions.

[0054] 1.2.2 GES-1 cell experiments

[0055] 1.2.2.1 Cell culture

[0056] The human gastric mucosal epithelial cell line was placed in DMEM (high glucose), 10% FBS and 1% P / S culture medium and cultured in a 37°C incubator with 5% CO2.

[0057] 1.2.2.2 Cytotoxicity assay

[0058] The experimental method of Zhang and Tang was modified to seed GES-1 cells in 96-well cell culture plates with 1×10 4 After the cells adhered to the wall, they were treated with different concentrations (0, 50, 100, 200, 400, 600, 800, 1000 μg / mL) of sea cucumber intestinal peptide for 24 hours. Then, MTT staining was added, and the crystals were dissolved by shaking with DMSO. The wavelength was selected at 570 nm, and the absorbance value of each well was measured. The cell survival rate was calculated by the absorbance of the treated cells and the control cells according to formula (2):

[0059] Survival rate (%) = (OD treatment group / OD control group) × 100 (2)

[0060] 1.2.2.3 Cell grouping and drug administration

[0061] Omeprazole and sea cucumber intestinal peptide were dissolved in 0.1% DMSO and sterile PBS to prepare mother solutions, and then diluted with DMEM high-glucose medium. GES-1 cells were divided into blank group, model group, omeprazole positive group (50 μg / mL) and sea cucumber intestinal peptide low-dose group (50 μg / mL), medium-dose group (100 μg / mL) and high-dose group (200 μg / mL). Six replicate wells were set in each group, and the edge wells of the culture plate were filled with sterile PBS, and a blank control group was set.

[0062] 1.2.2.4 Establishment of ethanol injury model

[0063] GES-1 cells in the logarithmic phase were selected, digested and counted, and seeded in 96-well cell culture plates, with 1×10 cells per well. 4 Cells were cultured in an incubator with 5% CO2 and 37°C for 24 hours to allow them to adhere to the wall. The blank group GES-1 cells were cultured in DMEM high-glucose incomplete medium, and the model group GES-1 cells were incubated in DMEM incomplete high-glucose medium containing alcohol (0, 3%, 4%, 5%, 6%, 7%, 8%, 9%) for 3 hours, and then the MTT experiment was performed to detect the activity of the cells. The experimental parameters when the cell survival inhibition rate was about 40% were taken as the best GES-1 injury model conditions. The inhibition rate (%) was calculated according to formula (3):

[0064] Inhibition rate (%) = (1-OD model group / OD control group) × 100 (3)

[0065] 1.2.2.5 MTT assay to detect cell proliferation

[0066] GES-1 cells in the logarithmic phase were selected, digested and counted, and seeded in 96-well cell culture plates, with 1×10 cells per well. 4 Cells were set up in 6 replicate wells in each group, and a blank control group was set up at the same time. They were cultured in an incubator with 5% CO2 and 37°C for 24 hours to allow them to adhere to the wall. Then, 6% anhydrous ethanol was used to induce for 3 hours to establish the injury model, and the drugs were divided and administered according to the ratio of 3.3.3.3, and incubated in an incubator with 37°C and 5% CO2 for 24 hours. The MTT method was used to detect cell activity, and the wavelength of 570nm was selected to measure the absorbance value of each group of cells, and the cell proliferation rate (%) was calculated according to formula (4):

[0067] Proliferation rate (%) = (OD sample group / OD blank group) × 100 (4)

[0068] 1.2.2.6 Detection of ROS production in GES-1 cells

[0069] Adjust the density of GES-1 cells to 1 × 10 5cfu / mL, and inoculated it on a 6-well plate with a density of 2mL per well. In a 37°C incubator, culture for 24 hours to ensure its adhesion, then use 6% anhydrous ethanol to induce for 3 hours to establish an injury model, and group the drugs according to the ratio of 3.3.3.3, incubate in an incubator at 37°C and 5% CO2 for 4 hours, and collect the cells. Incubate with incomplete medium containing 10μmol / L DCFH-DA for 30 minutes, invert it every 3 to 5 minutes to ensure that the probe and the cells can fully contact, wash the cells twice with serum-free cell culture medium, and then use flow cytometry to detect the generation of ROS in the cells, and then use a multifunctional microplate reader to measure the intracellular fluorescence intensity, set the excitation wavelength to 488nm and the emission wavelength to 525nm, and repeat 3 times for each group.

[0070] 1.2.2.7 Intracellular SOD and ADH enzyme activity detection

[0071] Adjust the density of GES-1 cells to 1 × 10 5 cfu / mL, and inoculated them on a 6-well plate with a density of 2mL per well. In a 37°C incubator, the original culture medium was discarded after culturing for 24 hours, and then 6% anhydrous ethanol was used to induce for 3 hours to establish an injury model. The drugs were divided and administered according to the ratio of 3.3.3.3, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The cells were collected and lysed, and the SOD enzyme activity, MDA content and total protein content in the supernatant were determined using SOD, MDA and BCA detection kits according to the instructions. The test results were expressed in U / mgprot, and each group was repeated 3 times.

[0072] 1.2.3 Protein purification

[0073] The sea cucumber intestinal peptide component with a molecular weight of less than 1KDa was purified using a Sephadex G-15 column. First, it was sterilized by filtration with a sterile filter membrane, and 2 mL was loaded. Deionized water was used as the mobile phase, the flow rate was 1 mL / min, the gradient elution was performed, the detection wavelength was 220 nm, and the absorption peaks were collected in sequence. After the collected components were freeze-dried, antioxidant tests were performed.

[0074] 1.2.4 De novo sequencing

[0075] 1.2.4.1 Sample pretreatment

[0076] The sample was dissolved in 50 mM NH4HCO3, and DTT solution was added to an appropriate amount of the sample to make the final concentration of 10 mmol / L. The sample was then reduced in a 56°C water bath for 1 h, and IAM solution was added to make the final concentration of 50 mmol / L. The sample was reacted in the dark for 40 min, and desalted using a self-filled desalting column. The solvent was evaporated in a 45°C vacuum centrifugal concentrator.

[0077] 1.2.4.2 Chromatographic conditions

[0078] Separation was carried out on an analytical column (150 μm i.d. × 150 mm), mobile phase A was 0.1% formic acid in water, mobile phase B was 80% ACN and 0.1% formic acid in water, gradient elution was from 4% to 95% in 66 min, flow rate was 600 nL / min.

[0079] Table 1 Liquid chromatography conditions

[0080]

[0081] 1.2.4.3 Mass spectrometry conditions

[0082] The liquid chromatograph (Easy-nLC 1200) was connected to an electrospray-ion trap-Orbitrap mass spectrometer (QExactive TM Hybrid Quadrupole-Orbitrap TM Mass Spectrometer). The primary mass spectrometer parameters are AGCtarget: 3e6, MaximumIT: 100ms, Resolution: 70000, Scanrange: 100to1500 m / z; the secondary mass spectrometer parameters are AGC target: 1e5, Resolution: 17500, TopN: 20, Maximum IT: 50ms, NCE / steppedNCE: 28.

[0083] 1.2.4.4 Mass spectrometry data analysis

[0084] The mass spectrometry raw files were analyzed for peptide sequences using the method of PEAKS Studio10.6 Denovo software. The retrieval parameters were as follows: the primary mass spectrometry error and the secondary mass spectrometry error were 20 ppm and 0.02 Da, respectively, the fixed modifications were Carbamidomethyl (C), the variable modifications were Oxidation (M) and Acetylation (N-term), the enzyme was Unspecific, and the maximum missed cleavage sites were 2.

[0085] 1.2.5 Bioinformatics and network pharmacology screening

[0086] 1.2.5.1 Active peptide screening

[0087] The peptide sequences identified by LC-MS / MS were submitted to a web-based peptide sequence analysis tool for activity screening. The molecular structures of the peptides were determined using ChemDraw 20.0 software.

[0088] 1.2.6 Toxicity and physicochemical properties of bioactive peptides released after simulated digestion

[0089] The BIOPEP-UWM database was used to perform computer-simulated gastrointestinal digestion of the most active peptides after screening; pepsin (EC 3.4.23.1), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) were used as representative digestive enzymes to perform simulated gastrointestinal digestion to study their potential resistance to pepsin in order to determine their digestive effects; the database ToxinPred was used to predict the potential toxicity of peptides released by simulated gastrointestinal digestion.

[0090] 2 Results and analysis

[0091] 2.1 Effect of single factor on hydrolysis degree

[0092] Temperature changes can affect the catalytic activity of enzymes1, such as Figure 1 As shown in a, from 40℃ to 60℃, the hydrolysis degree of sea cucumber intestinal polypeptide showed an upward trend, and the hydrolysis degree reached the highest at 60℃; when the temperature continued to rise, the structure of the enzyme was affected and began to denature, resulting in a decrease in the hydrolysis degree.

[0093] like Figure 1 As shown in Figure b, when the enzyme dosage is 4000U / g, the hydrolysis degree reaches the highest. When the enzyme dosage is reduced, the hydrolysis degree also decreases significantly due to insufficient catalytic activity of the enzyme. When the enzyme dosage is increased to 5000U / g, the hydrolysis degree does not change much. At this time, the substrate is close to saturation, and the contribution to the improvement of the hydrolysis degree is weakened. Increasing the enzyme dosage will cause a waste of production costs.

[0094] At the beginning of the reaction, there is a linear relationship between the amount of substrate and the number of active sites1. As the reaction proceeds, the number of unbound sites decreases over time. Therefore, Figure 1 As shown in Figure c, the degree of hydrolysis first increases and then decreases with the increase of enzymatic hydrolysis time. After 3.0 h, as the substrate is exhausted, over-enzymatic hydrolysis occurs, resulting in a decrease in the degree of hydrolysis.

[0095] 2.2 Response surface analysis

[0096] Based on the results of the single factor experiment, three variables, namely, enzymatic hydrolysis time (A), enzyme addition amount (B) and temperature (C), were selected to investigate their effects on the degree of hydrolysis. The response surface experimental design and results are shown in Table 2. The regression equation corresponding to the degree of hydrolysis is: DX (%) = 52.89-0.61A + 2.52B-2.10C + 0.42AB-0.065AC-1.40BC-2.60A 2 -3.14B 2 -5.19C 2 .

[0097] Analysis of variance (ANOVA) was used to estimate the significance of the fit of the second-order polynomial equation to the experimental data (Table 3). The quadratic regression model was significant (P < 0.05), while the lack-of-fit term was not significant (P > 0.05), indicating that the mathematical model in this study was reliable. The coefficient of determination (R 2 ＝ 0.9899), indicating that the model has good accuracy, and the adjusted determination coefficient (R 2 adj = 0.977), indicating that the experimental value is consistent with the predicted value. In addition, the coefficient of variation (CV) is less than 10%, indicating that the model has good accuracy and reproducibility, and the regression equation can well describe the relationship between each variable and the response value. According to the size of the F value, the influence of each variable on the test results is: B>C>A.

[0098] Table 2 Independent variable levels and Box-Behnken experimental design

[0099]

[0100]

[0101] Table 3 Results of variance analysis

[0102]

[0103] Note: *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: not significant. The steepness and slope of the surface can reflect the significance of the interaction between the factors. The 3D relationship diagram of each factor and hydrolysis degree is shown in Figure 2 As shown, the contour lines of temperature and time, temperature and enzyme addition, time and enzyme addition are all elliptical in shape and densely distributed. The slope and inclination of the curve are also very steep, indicating that the interactions between the factors are obvious and have a significant impact on the hydrolysis degree. The prediction of the interaction between the hydrolysis degree and the two combined variables shows extremely high accuracy, and the results are consistent with the regression model.

[0104] According to the response surface analysis results, the optimal hydrolysis conditions were obtained as follows: temperature 53.76℃, enzyme dosage 4453.63U / g, reaction time 2.96h, and the predicted maximum hydrolysis degree was 53.76%. Considering the convenience of actual operation, the optimal hydrolysis conditions were revised to: temperature 54℃, enzyme dosage 4454U / g, time 3h. Under these conditions, the degree of hydrolysis was 52.28%, and the relative error was 2.83% compared with the predicted value.

[0105] 2.3 In vitro antioxidant activity assay

[0106] Antioxidants have the function of preventing oxidative deterioration in food and preventing pathological processes mediated by oxidative stress in the human body, so they are attracting more and more attention. Figure 3 As shown, the lower the molecular weight, the stronger the antioxidant activity, and the intensity is P3>P2>P1.

[0107] The most commonly used method to determine the DPPH free radical scavenging rate is to determine the antioxidant capacity based on electron transfer. DPPH solution has a strong absorption capacity in the ultraviolet visible light region. When the free radicals encounter antioxidants, they are scavenged and the absorbance decreases. Figure 3 In a, as the concentration increases, the DPPH free radical scavenging rates of sea cucumber intestinal polypeptides of different molecular weights all increase to varying degrees, showing a certain concentration dependence. When the concentration is 25 mg / mL, the P3 scavenging rate reaches 91.37%, showing strong DPPH free radical scavenging activity.

[0108] Superoxide anion radicals are first formed in cellular oxidation reactions. Although they are not highly reactive, they can produce hydrogen peroxide and hydroxyl radicals through dismutation and other types of reactions. They are the source of free radicals in the body, and their derivatives also cause damage to DNA and cell membranes. Figure 3 In b, the superoxide anion radical inhibition rate of sea cucumber intestinal polypeptides with different molecular weights increased with the increase of concentration. When the concentration was 25 mg / L, P3 showed strong activity and the inhibition rate reached 90.42%.

[0109] Redox is an essential metabolic reaction in biological systems. Antioxidants give out electrons through their own reduction, and free radicals receive electrons to form stable products, thereby blocking the chain reaction of the oxidation process. Hydroxyl free radicals are the most aggressive and dangerous incomplete oxygen reduction products in the body, which can easily cause serious damage to surrounding biological molecules, causing cancer, aging, etc. Figure 3 In c, as the concentration increases, the scavenging rates of P2 and P3 hydroxyl radicals gradually increase, and the activity of P1 does not change much. When the concentration is 25 mg / L, the scavenging rate of P3 hydroxyl radicals reaches 90.99%.

[0110] 2.4 Determination of the ability of sea cucumber intestinal peptides to protect and repair gastric mucosa

[0111] 2.4.1 Establishment of ethanol-induced GES-1 cell injury model

[0112] Ethanol can induce cell dehydration, degeneration and necrosis. Currently, it is a common method to use ethanol to induce gastric mucosal injury model. Figure 4 As shown in the figure, when different concentrations of alcohol acted on GES-1 cells for 3 hours, different degrees of damage were caused to GES-1, and the cell activity decreased significantly with the increase of concentration. Among them, the inhibition rate of 6% alcohol incubation for 3 hours was 41.51%. This parameter is the optimal condition for establishing the GES-1 cell damage model.

[0113] 2.4.2 Effects of sea cucumber intestinal peptide on GES-1 cell activity

[0114] Effects of sea cucumber intestinal peptide on the activity of GES-1 cells damaged by ethanol, such as Figure 5 As shown, after sea cucumber intestinal peptide acted on normal GES-1 cells for 24 hours, it had no significant effect on the activity of GES-1 cells in the concentration range of 50-200 μg / mL compared with the control group; when the concentration reached 400 μg / mL, the activity of the cells was significantly inhibited (P<0.01). If the concentration of the sample is further increased, the activity of the cells will be extremely significantly inhibited (P<0.001). Therefore, samples with concentrations of 50, 100, and 200 μg / mL were selected as the low-dose group, medium-dose group, and high-dose group of sea cucumber intestinal peptide.

[0115] 2.4.3 Effect of sea cucumber intestinal peptide on the proliferation of ethanol-damaged GES-1 cells

[0116] Effects of sea cucumber intestinal peptide on the proliferation of GES-1 cells damaged by ethanol, such as Figure 6 As shown. Compared with the blank group, the proliferation of GES-1 cells in the model group decreased significantly (P<0.001). Compared with the model group, the model group treated with sea cucumber intestinal peptides had a good protective effect on GES-1 cells, and the cell viability showed concentration dependence, that is, as the concentration of sea cucumber intestinal peptides increased, their protective effect on cells increased. The sea cucumber intestinal peptides in the three dose groups significantly increased the proliferation ability of GES-1 cells (P<0.001), among which the cell proliferation ability of the high dose group was not significantly different from that of the control group.

[0117] 2.4.4 Effect of sea cucumber intestinal peptide on ROS clearance in ethanol-damaged GES-1 cells

[0118] Reactive oxygen species (ROS) are composed of hydroxyl radicals, superoxide anions, hydrogen peroxide, ozone, and singlet oxygen. Under normal circumstances, it is maintained at a relatively low level in the body and participates in the signaling process of cells. However, when the body's antioxidant defense mechanism cannot completely eliminate these reactive oxygen species, oxidative stress may be induced. When cells are subjected to oxidative stress, intracellular ROS will increase significantly, and the concentration of intracellular ROS can intuitively reflect the degree of damage to the cells. Excessive intake of anhydrous ethanol may cause acute gastric mucosal damage and trigger oxidative stress. The intake of anhydrous ethanol may weaken the antioxidant function of cells, thereby inducing oxidative stress. In the process of ethanol triggering the body's stress response, ROS, as a key stimulus signal, plays a vital role in the entire process of gastric mucosal damage.

[0119] In this study, we detected ROS levels as a marker of oxidative stress. The effect of sea cucumber intestinal peptide on the ROS content in GES-1 cells was detected by flow cytometry under the FL1-A channel. The horizontal axis of the image is the fluorescence channel, and the vertical axis is the number of cells. Figure 7 As shown, the positive control group and the three dose groups can significantly reduce the level of ROS in damaged GES-1 cells in a concentration-dependent manner. These results indicate that sea cucumber intestinal peptide can show a good protective effect by regulating the level of oxidative stress in gastric mucosal cells damaged by ethanol.

[0120] 2.4.5 Effect of sea cucumber intestinal peptide on SOD activity in ethanol-damaged GES-1 cells

[0121] Superoxide anion is a type of reactive oxygen species, which is produced by single electron reduction of oxygen molecules in cells. It can diffuse through cells and play a key role in various physiological processes such as cell growth and cell death, especially in the redox metabolism of cells. In cells, superoxide anions can be converted into H2O2 by superoxide dismutase. However, when the superoxide anions produced in an organism exceed the regulatory capacity of the cell, it may cause problems such as cell death, DNA damage and lipid peroxidation, which may lead to the emergence of chronic diseases such as neurodegenerative diseases and diabetes. Superoxide dismutase constitutes the first defense layer of the organism's oxygen free radical scavenging mechanism and has the ability to repair cells. When the content of oxygen free radicals in cells increases, it can induce SOD biosynthesis and activity. Therefore, the level of SOD content can indirectly reflect the generation of oxygen free radicals and the ability of the organism to scavenge oxygen free radicals.

[0122] like Figure 8As shown in the results, we found that in the ethanol-induced GES-1 cell gastric mucosal injury model, the SOD activity was significantly reduced, and the sea cucumber intestinal peptide dose groups with different mass concentrations could significantly increase the SOD level, and showed a concentration-dependency; among them, there was no significant difference between the high-dose sea cucumber intestinal peptide group and the positive control group, which indicates that sea cucumber intestinal peptide can protect and repair the gastric mucosa by increasing the SOD level of damaged cells.

[0123] 2.4.6 Effect of sea cucumber intestinal peptide on MDA content in ethanol-damaged GES-1 cells

[0124] MDA is a type of lipid peroxide. It is the final product of long-chain fatty acids after oxidation. MDA can be used as a marker of oxidation, and changes in its content can indirectly reflect the degree of damage caused by oxygen free radicals. Fig. 9 As shown in the figure, compared with the control group, the MDA content in the model group increased significantly, and different concentrations of sea cucumber intestinal peptides could reduce the damage to cells and showed a concentration-dependency, which indicated that sea cucumber intestinal peptides could protect and repair gastric mucosa by reducing the MDA level of damaged cells.

[0125] 2.5 Antioxidant activity of purified fractions

[0126] Separation of sea cucumber enteric peptides (MW < 1 KDa) was performed using Sephadex G-15. Fig.10 As shown in Figure a, after elution with deionized water, 6 different elution components were obtained, which are T1-T6. According to the order of elution, the relative molecular weight of the eluted components is ranked from high to low: T1>T2>T3>T4>T5>T6. Among them, T6 has the slowest elution speed, which means that its relative molecular weight is the lowest. The antioxidant activity of the 6 components after elution was evaluated, as shown in Figure 1. Fig.10 As shown in Figures b, c, and d, all six eluted components showed antioxidant properties, among which the antioxidant activity of component T4 was more prominent. Therefore, T4 was selected for de novo sequencing analysis and further research.

[0127] 2.6 Active peptide screening

[0128] Bioinformatics is a discipline that uses computer technology to explore the laws of biological systems. Its research content and conclusions are a variety of biological data. With the rapid advancement of bioinformatics-based peptide screening methods, a series of databases and bioinformatics tools specifically for bioactive peptides have been developed. These tools provide detailed information about the sequence, structure, interaction between organisms, and diseases of peptides, which helps to better understand the physiological effects of bioactive peptides.

[0129] In this experiment, 91 sequences were obtained by LC-MS / MS sequencing of the T4 component, of which the molecular weight was distributed in the range of 400Da-1400Da and consisted of 4-14 amino acids. The 91 peptide sequences were submitted to the web-based active peptide analysis tool database, and the threshold was set to be greater than 0.8. As shown in Table 4, a total of 3 peptide sequences (WRPPPP, SHFFR, LPPR) were obtained, and their secondary structure mass spectra are shown in Figure 11-13 shown.

[0130] Table 4 Screening of sea cucumber intestinal peptide sequences

[0131]

[0132] 2.7 Toxicity of bioactive peptides released after simulated digestion

[0133] ToxinPred is a platform for evaluating the actual toxicity of peptides. It uses a model developed by the support vector machine (SVM) based on Swiss-Prot's machine learning technology to predict the toxicity of peptides released by simulated gastrointestinal digestion. Toxicity analysis prediction shows that the peptides released by simulated digestion of sea cucumber intestinal peptides are not potentially toxic, and the molecular weight is between 242.25-406.52Da.

[0134] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A sea cucumber intestinal polypeptide, characterized in that: Its amino acid sequence is one or more of those shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.

3.

2. Use of the sea cucumber intestinal polypeptide as claimed in claim 1 in the preparation of drugs for treating and / or preventing gastric diseases.

3. The use of the sea cucumber intestinal polypeptide according to claim 2 in the preparation of a drug for treating and / or preventing gastric diseases, characterized in that: The gastric disease is a gastric disease caused by damage to the gastric mucosa.

4. A pharmaceutical composition for treating and / or preventing gastric diseases, characterized in that: The pharmaceutical composition comprises the sea cucumber intestinal polypeptide or a pharmaceutically acceptable salt of the sea cucumber intestinal polypeptide as claimed in claim 1.

5. The pharmaceutical composition for treating and / or preventing gastric diseases according to claim 4, characterized in that: The pharmaceutical composition further comprises one or more therapeutic agents; the therapeutic agents include drugs having a protective effect on the gastric mucosa.

6. The pharmaceutical composition for treating and / or preventing gastric diseases according to claim 4, characterized in that: Its components also include at least one of pharmaceutically acceptable carriers, excipients, adjuvants and vehicles.

7. The pharmaceutical composition for treating and / or preventing gastric diseases according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is an oral preparation, a mucosal administration preparation or an injection.

8. The pharmaceutical composition for treating and / or preventing gastric diseases according to claim 4, characterized in that: The pharmaceutical composition is used for protecting and repairing gastric mucosal damage.

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