Application of cow placenta polypeptide in preparation of medicine for protecting intestinal injury caused by cyclophosphamide

Abstract

The invention discloses an application of cow placenta polypeptide in preparation of a medicine for protecting intestinal injury caused by cyclophosphamide, which comprises the following steps: cutting the placenta of a healthy Chinese Holstein cow in normal delivery into pieces, homogenizing by using a tissue homogenizer, adding papain, acting for 4.7 hours under the conditions that the pH value is 6.5 and the temperature is 55 DEG C, centrifuging after inactivating for 10 minutes in a boiling water bath, collecting the supernatant, and performing freeze-drying to obtain the cow placenta polypeptide. The dairy cow placenta polypeptide ANNGKQWAEVF (H-Ala-Asn-Asn-Gly-Lys-Gln-Trp-Ala-Glu-Val-Phe-OH) is screened through a virtual screening method and molecular docking after freeze drying, the affinity of the dairy cow placenta polypeptide ANNGKQWAEVF (H-Ala-Asn-Asn-Gly-Lys-Gln-Trp-Ala-Glu-Val-OH) and Keap1 is the strongest, the molecular formula of the dairy cow placenta polypeptide is C57H82N16O17, and the molecular weight of the dairy cow placenta polypeptide is 1263.38 Da. The dairy cow placenta polypeptide can improve the villus form of the Release of intestinal barrier related factors is regulated, and expression of intestinal barrier genes is up-regulated.

Description

Technical Field

[0001] The invention relates to the technical field of drug extraction, and in particular to use of a cow placenta extract in preparing a drug for protecting intestinal damage caused by cyclophosphamide. Background Art

[0002] Cyclophosphamide (Cy) is widely used as a chemotherapy drug, which can cause a series of pathological changes such as changes in intestinal mucosal morphology, induction of oxidative damage and immune dysfunction, and ultimately lead to intestinal dysfunction. Placental tissue contains a large number of bioactive substances such as polypeptides, cyclic peptides, steroids, collagen and amino acids, which have the effects of anti-oxidation, anti-fatigue, promoting metabolism, enhancing physiological functions and improving immunity. The traditional clinical utilization method of traditional Chinese medicine is to collect fresh placenta, steam and dry it, and grind it into medicine. In recent years, researchers have effectively improved the biological activity and total substance content of placental products by changing the steaming conditions and increasing the processing materials, but placental polypeptides with relatively simple ingredients have not yet been obtained. In addition, there is no report on cow placental polypeptides as protective drugs for cyclophosphamide-induced intestinal damage in mice. Therefore, the present invention separates cow placental enzymatic hydrolyzate placental polypeptides by virtual screening method, and finds that the cow placental polypeptide molecular structure that can protect the intestinal damage caused by cyclophosphamide. Summary of the invention

[0003] To achieve the above object, the technical problem to be solved by the present invention is to provide a use of a cow placenta polypeptide as shown in formula I in the preparation of a drug for protecting intestinal damage caused by cyclophosphamide;

[0004]

[0005] To achieve the above object, the preparation of the cow placenta polypeptide of the present invention comprises the following steps:

[0006] Step 1, after the dairy cow placenta is cut into pieces, it is homogenized by a tissue homogenizer, papain is added, and the mixture is subjected to the conditions of pH 6.5 and temperature 55°C for 4.7 hours, inactivated in a boiling water bath for 10 minutes, and then centrifuged, the supernatant is collected, and freeze-dried to prepare a placental hydrolysate lyophilized powder; the proteins and peptides in the placental hydrolysate lyophilized powder are identified by using LC-MS / MS technology: the sample is separated by using the HPLC liquid phase system Easy-nLC with a nanoliter flow rate, and then the amino acid sequence of the polypeptides in the placental hydrolysate lyophilized powder is analyzed by mass spectrometry using a Q Exactive mass spectrometer;

[0007] Step 2: The amino acid sequence of the polypeptides in the placental hydrolysate lyophilized powder obtained in step 1 was predicted by AnOxPP (http: / / www.cqudfbp.net / AnOxPP / index.jsp), ProtParam (http: / / we-b.expasy.org / protparam / ), Innovagen (http: / / www.innovagen.com / proteomics-tools / ), ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / ) and AllerTOP (v.2.0) (https: / / www.ddgpharmfac.net / AllerTOP / ) to predict their antioxidant activity, stability, water solubility, toxicity and sensitization, and the top 5 polypeptides were screened as potential bovine placental polypeptides.

[0008] Step 3: Further screening of bovine placental peptides using molecular docking method; first, the 3D structure of potential CPAP was drawn using ChemDraw19.0 software, imported into Discovery Studio 2019 client software, removed hydrogen atoms as ligands, and TX6 (Pub Chem ID: 121488089) was selected as a positive control; secondly, the Keap1 protein crystal structure (PDB ID: 2FLU) was downloaded from the RCSB protein database (https: / / www.rcsb.org), imported into DiscoveryStudio2019 client software, removed ligands and water, added hydrogen and cleaned the protein, and used as a receptor; then, the active center (x: 5, y: 9, z: 1, radius: ), and docking was performed using the "-CDOCKER" protocol; finally, the bovine placenta peptides were screened based on the -CDOCKER_INTE-RACTION_ENERGY (-CIE) score after docking;

[0009] Step 4: Based on the amino acid sequence of the cow placenta polypeptide, it can be synthesized by solid phase method with a purity greater than 95%;

[0010] A cow placenta polypeptide of the present invention is ANNGKQWAEVF (H-Ala-Asn-Asn-Gly-Lys-Gln-Trp-Ala-Glu-Val-Phe-OH), and its molecular formula is C 57 H 82 N 16 O 17 , with a molecular weight of 1263.38Da, can be used as a drug to protect intestinal damage caused by cyclophosphamide.

[0011] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0012] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the CPE LC-MS / MS detection result.

[0014] Figure 2 This is the interaction between the cow placental polypeptide ANNGKQWAEVF and Keap1 (PDB ID: 2FLU).

[0015] Figure 3 This is the liquid chromatography and secondary mass spectrometry of cow placenta peptides.

[0016] Figure 4 These are the test results of body weight and immune organ index of experimental mice.

[0017] Figure 5 These are the test results of serum DAO and D-LA in experimental mice.

[0018] Figure 6 These are the test results of the intestinal villi tissue structure of experimental mice.

[0019] Figure 7 These are the test results of sIgA and β-DF levels in the intestine of experimental mice.

[0020] Figure 8 These are the test results of intestinal immune-related cytokines in experimental mice.

[0021] Fig. 9 These are the test results of intestinal oxidation indicators in experimental mice.

[0022] Fig.10 These are the results of relative mRNA expression of intestinal barrier-related genes in experimental mice. DETAILED DESCRIPTION

[0023] Example 1. Preparation of cow placenta polypeptide

[0024] 1 Materials and methods

[0025] 1.1 Materials and Instruments

[0026] 1.1.1 Materials and reagents

[0027] Cow placenta was collected from healthy cows that gave birth naturally in large-scale ranches; 30DEAE cellulose ion exchange resin filler (L10W08KB), Nanovita Technology Co., Ltd.; Sephadex G25 gel filler (RH314160), Shanghai Roen Chemical Technology Co., Ltd.; sodium bicarbonate (144-55-8), sodium hydroxide (1310-73-2), sodium chloride (7647-14-5), Chengdu Cologne Chemical Co., Ltd.; hydrochloric acid (7647-01-0), Xilong Chemical Co., Ltd.; phosphate buffer (ST448), Shanghai Biyuntian Biotechnology Co., Ltd.; papain (G8430), Solarbio, USA; DPPH micro kit (R27137), Shanghai Yuanye Biotechnology Co., Ltd.; formic acid (695076) and acetonitrile (34851), Sigma, USA.

[0028] 1.1.2 Test instruments and equipment

[0029] Pen pH meter (PH828), Dongguan Wanchuang Electronic Products Co., Ltd.; tissue homogenizer (FSH-2A), Changzhou Yuexin Instrument Manufacturing Co., Ltd.; electronic balance (JM-A30002), Zhuji Chaoze Weighing Equipment Co., Ltd.; constant temperature water bath (HH-S4A), Beijing Kewei Yongxing Instrument Co., Ltd.; vacuum freeze dryer (SCIENTZ-10N-A), Ningbo Xinzhi Freeze Drying Equipment Co., Ltd.; desktop high-speed refrigerated centrifuge (TGL-16.5M), Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.; high performance liquid chromatograph (Waters2695), Waters Company, USA; ultra-micro spectrophotometer (Nano Drop OneC), high performance liquid chromatograph (Easy-nLC 1200), mass spectrometer (Q Exactive combined quadrupole Orbitrap), multi-function fluorescence chemiluminescence analyzer (Varioskan Flash), Thermo Fisher Scientific, USA.

[0030] 1.2 Test methods

[0031] 1.2.1 Preparation of cow placenta extract

[0032] The cow placenta was cut into pieces and homogenized with a tissue homogenizer, and papain was added. It was incubated at pH 6.5 and 55°C for 4.7 hours, inactivated in a boiling water bath for 10 minutes, and then centrifuged. The supernatant was collected and freeze-dried to make placental enzymatic hydrolysate lyophilized powder. The proteins and peptides in the placental enzymatic hydrolysate lyophilized powder were identified using LC-MS / MS technology: the sample was separated using the HPLC liquid phase system Easy-nLC with a nanoliter flow rate, and then the mass spectrometry analysis was performed using a Q Exactive mass spectrometer, which was repeated three times.

[0033] 1.2.2 Isolation of bovine placental peptides using virtual screening and chromatography

[0034] (1) Virtual screening method to isolate bovine placental peptides

[0035] The amino acid sequences of peptides in the placental hydrolysate lyophilized powder were predicted by AnOxPP (http: / / www.cqudfbp.net / AnOxPP / index.jsp), ProtParam (http: / / we-b.expasy.org / protparam / ), Innovagen (http: / / www.innovagen.com / proteomics-tools / ), ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / ) and AllerTOP (v.2.0) (https: / / www.ddgpharmfac.net / AllerTOP / ) to predict their antioxidant activity, stability, water solubility, toxicity and allergenicity, and the top five peptides were screened as potential bovine placental peptides.

[0036] Molecular docking was used to further screen bovine placental peptides. First, the 3D structure of potential bovine placental peptides was drawn using ChemDraw19.0 software, imported into Discovery Studio 2019 client software, and used as a ligand after removing hydrogen atoms, and TX6 (Pub Chem ID: 121488089) was selected as a positive control. Secondly, the Keap1 protein crystal structure (PDB ID: 2FLU) was downloaded from the RCSB protein database (https: / / www.rcsb.org), imported into DiscoveryStudio2019 client software, and used as a receptor after removing ligands and water, adding hydrogen, and cleaning the protein. After that, the active center (x: 5, y: 9, z: 1, radius: ), and docking was performed using the "-CDOCKER" protocol. Finally, the cow placenta peptides were screened based on the -CDOCKER_INTE-RACTION_ENERGY (-CIE) score after docking.

[0037] 1.2.3 Synthetic cow placenta peptide

[0038] It is synthesized according to the amino acid sequence of cow placenta polypeptide by solid phase method, with a purity of more than 95%.

[0039] 1.3 Data Analysis

[0040] SPSS26.0 software was used for statistical analysis, and the mean ± standard deviation of each index was calculated. Dunnett's T3 method was used for significance analysis. P>0.05 indicated no significant difference, P<0.05 indicated significant difference, and P<0.01 indicated extremely significant difference. GraphPad Prism 9.0.0 software was used for drawing.

[0041] 2 Results and analysis

[0042] 2.1 Components of placenta hydrolysate freeze-dried powder

[0043] The results of LC-MS / MS test of placental enzymatic hydrolysate lyophilized powder are as follows Figure 1 As shown in the figure, the peaks of the three placental hydrolysate freeze-dried powder samples all appeared below 400m / z, and the waveforms were similar, indicating that the placental hydrolysate freeze-dried powder preparation method used had good repeatability. After removing duplicates, a total of 128 peptides were determined, with relative molecular weights below 3000u, peptide lengths concentrated in 7-25, and derived from 85 proteins.

[0044] 2.1 Virtual screening method to isolate bovine placental peptides

[0045] The placental hydrolysate freeze-dried powder contains a total of 128 peptides. After the prediction of antioxidant activity, water solubility, stability, sensitization and toxicity, it was found that 68.75% of the peptides had antioxidant activity, 43.75% of the peptides had good stability, 68.75% of the peptides had good water solubility, 53.91% of the peptides were non-sensitizing, and 100% of the peptides were non-toxic. The top five peptides in terms of physicochemical properties were screened out as potential bovine placental peptides (as shown in Table 1) for molecular docking. By comparing with the known functional bioactive peptides in the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), it was found that these five peptides were all new bioactive peptides.

[0046] Table 1 Prediction results of peptide physicochemical properties

[0047]

[0048] Molecular docking can be used to determine the binding affinity of cow placental peptides to Keap1 and screen cow placental peptides. As shown in Table 2, the -CIE values ​​of the five peptides are 109.87, 109.32, 105.85, 105.81, and 51.94 kJ / mol, respectively, which are all higher than the -CIE value of the positive control TX6 (25.58 kJ / mol), indicating that the affinity of the five peptides to Keap1 is higher than that of TX6, and ANNGKQWAEVF has the strongest affinity. Figure 2It can be seen that ANNGKQWAEVF can compete with Nrf2 for binding sites by occupying the area above the central cavity of the Keap1 Kelch domain ( Figure 2 A, docking 3D structure diagram); ANNGKQWAEVF forms hydrogen bond interactions through 13 amino acid residues including TYR334, ALA366, ARG380, forms hydrophobic interactions with LEU557, IE559, TYR572 amino acid residues, forms electrostatic interactions with ARG415 amino acid residues, and occupies 3 key sites ( Figure 2 B, docking 2D structure diagram), indicating that ANNGKQWAEVF is stably bound to Keap1. Therefore, ANNGKQWAEVF (H-Ala-Asn-Asn-Gly-Lys-Gln-Trp-Ala-Glu-Val-Phe-OH) was selected, and its molecular formula is C 57 H 82 N 16 O 17 , molecular weight is 1263.38Da.

[0049] Table 2 Peptide molecular docking results

[0050]

[0051]

[0052] 2.2 Purity analysis of synthetic cow placenta peptides

[0053] After solid phase synthesis of cow placental peptides, its liquid chromatography and mass spectrometry are as follows Figure 3 As shown, the purity is greater than 95%.

[0054] Example 2: Use of cow placenta polypeptide in medicine to protect intestinal damage caused by cyclophosphamide

[0055] 1. Materials and methods

[0056] 1.1 Materials and Instruments

[0057] 1.1.1 Experimental animals

[0058] Forty-eight 8-week-old male Kunming mice, weighing 43.75 ± 0.19 g, were purchased from Beijing Sibeifu and fed with a standard diet (production license number: SCXK (Chuan) 2019-028; production standard: GB14924·3-2010).

[0059] 1.1.2 Main test reagents and kits

[0060] Cow placenta polypeptide; 4% paraformaldehyde (20220805), Shanghai Biyuntian; cyclophosphamide (S30563), Shanghai Rui'an; isoflurane (26675467), Shandong Yihong; HE staining kit (G1003), RNA extraction solution (G3013), cDNA synthesis kit (G3330) were all purchased from Wuhan Saiweier; DAO (20220391), D-LA (2022211), SOD (20223 34), GSH-Px (20222335), CAT (20222435), MDA (20222112), TNF-α (2022344), IL-1β (20224532), IL-4 (2022345), IL-10 (20223445), sIgA (20223454), and β-DF (202224567) ELISA kits were purchased from Shanghai ELISA.

[0061] 1.1.3 Main test instruments

[0062] Ultra-low temperature refrigerator (DW-86L828W), Haier; electronic balance (JM-A30002), Zhuji Chaoze Scale; desktop centrifuge (TGLB-16G), Sigma, Germany; multi-function fluorescence chemiluminescence immunoassay (Varioskan Flash), Thermo Fisher Scientific, USA; fluorescence quantitative PCR instrument (CFX), Shanghai Bio-Rad; paraffin sectioner (RM2235), Leica, Germany; upright optical microscope (NikonEclipse E100), imaging system (DS-U3), Nikon, Japan.

[0063] 1.2 Test methods

[0064] 1.2.1 Experimental mouse grouping and model establishment

[0065] After 7 days of adaptive feeding, 48 mice were randomly divided into 4 groups, 12 mice per group. The control group (group C), the healthy placenta extract group (group H), the model group (group M), and the model intervention group (group T). Groups C and M were fed with a standard diet, and groups H and T were fed with a standard diet + 1500 mg / kg / d of cow placenta peptide (the preliminary test results showed that among the four concentration gradients of 500 mg / kg, 1000 mg / kg, 1500 mg / kg, and 2000 mg / kg, 1500 mg / kg had the most significant effect on the improvement of intestinal sIgA levels, so this dose was selected as the formal test additive dose), once a day for 28 consecutive days. On the 26th day of the experiment, mice in groups M and T were intraperitoneally injected with cyclophosphamide (80 mg / kg), and groups C and H were intraperitoneally injected with an equal volume of saline, once a day for 3 consecutive days. The body weight of mice was measured every week.

[0066] Standards for establishing an intestinal injury model: mice develop symptoms such as unkempt hair, depression, reduced movement and weight loss; the thymus and spleen atrophy, and a decrease in the immune organ index; intestinal sections show that the villi structure is destroyed, with varying lengths and sparse arrangement.

[0067] 1.2.2 Sample collection and storage

[0068] (1) Serum collection and storage: After the experiment, 12 mice in each group were fasted for 12 hours, and the eyeballs were removed under isoflurane anesthesia for blood collection. The blood was placed in a centrifuge tube without anticoagulant, left to stand at room temperature for half an hour, and centrifuged at 4°C, 6000 rpm / min for 5 minutes to obtain serum, which was numbered and stored in a -80°C refrigerator.

[0069] (2) Immune organ collection: Thymus and spleen were collected from 12 mice in each group, fascia and fat were removed, excess water was absorbed with filter paper and weighed for calculation of immune organ index. Immune organ index (mg / g) = immune organ weight (mg) / body weight (g).

[0070] (3) Small intestinal tissue collection and preservation: Three mice were randomly selected from each group, and one sample of the duodenum, jejunum and ileum was collected respectively. The samples were rinsed with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde solution, and numbered for small intestinal mucosal morphological observation. The mid-section jejunum tissue samples were collected from each group of mice, rinsed with PBS, numbered, and stored in a -80°C refrigerator for the preparation of intestinal homogenate. Six mice were randomly selected from each group, and the mid-section jejunum tissue samples were collected. The samples were numbered, quickly frozen in liquid nitrogen, placed in enzyme-free tubes, and stored in a -80°C refrigerator for gene expression detection.

[0071] 1.3 Index determination

[0072] 1.3.1 Serum index determination

[0073] The DAO activity and D-LA level in each serum sample were determined according to the kit instructions.

[0074] 1.3.2 Morphological determination of small intestinal mucosa

[0075] Embedding: The duodenum, jejunum and ileum tissue samples fixed with 4% paraformaldehyde were rinsed in running water for 30 minutes, and then the tissue blocks were trimmed and dehydrated with ethanol from low to high concentrations. The tissue blocks were then placed in xylene to make them transparent, and then the transparent tissue blocks were embedded in paraffin.

[0076] Sectioning: Use a microtome to cut the tissue block into 5 μm thick slices, flatten them in warm water, place them on a slide, and bake them at 60°C for more than 2 h.

[0077] H&E staining: After the sections were dewaxed with xylene, they were rinsed with running water for 20 minutes, stained with hematoxylin for 30 minutes, rinsed with running water for 20 minutes, differentiated with hydrochloric acid and alcohol, stained with eosin for 5 minutes, and finally dehydrated with gradient alcohol. After being transparentized with xylene, the sections were sealed with resin glue.

[0078] Images were taken using an optical microscope at 100× magnification, and Image Pro Plus 6.0 was used to measure the height of the villi and the corresponding crypt depth in the tissues, and the ratio of the villus height to the crypt depth was calculated.

[0079] 1.3.3 Determination of related indicators of jejunum tissue

[0080] Weigh the jejunal tissue, add PBS and homogenize thoroughly in a glass homogenizer (weight: volume = 1:9), centrifuge at 8000r / min at 4℃ for 20min, and collect the tissue supernatant. Determine the activity and level of sIgA, β-DF, TNF-α, IL-1β, IL-4, IL-10, SOD, MAD, GSH-Px, and CAT according to the instructions of the kit.

[0081] 1.3.4 Detection of relative expression of intestinal barrier gene mRNA

[0082] Total RNA was extracted from jejunal tissue using TRlzol RNA extraction reagent, cDNA was obtained using FastQuant RT Kit reverse transcription kit, primers were designed using primer5 (Table 3), and real-time fluorescence quantitative PCR was performed using GAPDH as the internal reference gene. -ΔΔCT Method for analysis.

[0083] Table 3 Primer sequences and parameters

[0084]

[0085] 1.4 Statistical analysis

[0086] The test results are expressed as mean ± standard error. SPSS26.0 software was used for data analysis. One-way ANOVA was used for multiple comparisons between groups, followed by Dunnet analysis. P<0.05 was considered significant, and P<0.01 was considered extremely significant. GraphPad Prism9.0 was used for drawing. Table: Superscript letters indicate differences between groups, subscript letters indicate differences within groups, the same letters indicate no significant differences P>0.05, and different letters indicate significant differences P<0.05. Figure: "*" indicates significant differences; "**" indicates extremely significant differences; no letters between groups indicate no significant differences.

[0087] 2 Test results

[0088] 2.1 Results of test mouse body weight and immune organ index

[0089] During the entire test period, no mice in each group showed obvious poisoning or death. Figure 4 There was no significant difference in the body weight of mice in the first 21 days (P>0.05). On the 28th day, the body weight of group M was significantly lower than that of groups C and H (P<0.01), and lower than that of group T (P>0.05). There was no significant difference in body weight among groups C, H, and T. The thymus and spleen indexes of group M were significantly lower than those of group C (P<0.01). The spleen and thymus indexes of group H were higher than those of group C (P>0.05). The thymus index of group T was significantly higher than that of group M (P<0.05), and the spleen index was higher (P>0.05), and there was no significant difference between them and group C. This indicates that bovine placental polypeptides can alleviate the damage of Cy to mice by increasing the body weight and immune organ index of mice, and will not cause adverse effects on healthy mice.

[0090] Table 4 Changes in mouse body weight during the experiment

[0091]

[0092] 2.2 Results of serum DAO and D-LA in experimental mice

[0093] like Figure 5 Compared with group C, the DAO activity and D-LA level in group M were significantly higher (P<0.01). Compared with group C, the DAO activity and D-LA level in group H were lower (P>0.01). Compared with group M, the DAO activity in group T was significantly lower (P<0.05), and the D-LA level was significantly lower (P<0.01), with no significant difference from group C. This indicates that cow placental polypeptide can reduce the integrity and damage of the intestinal mechanical barrier caused by Cy, and will not damage the intestinal barrier of healthy mice.

[0094] 2.3 Results of intestinal mucosal morphology test in experimental mice

[0095] like Figure 6 The villi of group C were intact, with neat and tight arrangement and clear boundaries; the villi of group M were damaged, with different lengths and sparse arrangement; the villi of group H were more complete than those of group C; the villi of group T were improved to varying degrees compared with those of group M. In addition, the height of duodenal villi, the height of jejunal villi, and the ratio of duodenal villus to crypt in group M were significantly decreased compared with those of group C (P<0.01), and the depth of ileal crypt was significantly increased (P<0.05). The height of villi in group H was increased compared with group C, and the depth of crypt was decreased compared with group M (P>0.05). This indicates that bovine placental polypeptides can maintain the morphological structure of intestinal mucosa by repairing damaged intestinal villi.

[0096] 2.4 Results of intestinal sIgA and β-DF detection in experimental mice

[0097] like Figure 7 Compared with group C, the sIgA level in group M was significantly lower (P<0.01), and the β-DF level was significantly lower (P<0.05). Compared with group C, the sIgA and β-DF levels in group H were significantly higher (P<0.05). Compared with group M, the sIgA level in group T was significantly higher (P<0.01), and the β-DF level was higher (P>0.05), and there was no significant difference between these two groups. This indicates that bovine placental polypeptides can enhance the intestinal barrier function of mice with intestinal damage caused by Cy and strengthen the intestinal immune defense ability.

[0098] 2.5 Results of intestinal immune-related cytokine detection in experimental mice

[0099] like Figure 8 Compared with group C, the levels of TNF-α, IL-1β, IL-4, and IL-10 in group M were significantly lower (P<0.01). Compared with group C, the levels of TNF-α, IL-1β, IL-4, and IL-10 in group H were higher (P>0.05). Compared with group M, the levels of TNF-α and IL-10 in group T were significantly higher (P<0.01), the level of IL-4 was significantly higher (P<0.05), and the level of IL-1β was higher (P>0.05). Compared with group C, the levels of TNF-α, IL-1β, and IL-4 in group T were significantly lower (P<0.01), and the level of IL-10 was significantly lower (P<0.05). This indicates that bovine placental polypeptides restore intestinal immune function by increasing the release of intestinal immune-related cytokines.

[0100] 2.6 Results of intestinal oxidation index test in experimental mice

[0101] like Fig. 9 Compared with group C, the MDA level in group M was significantly higher (P<0.01), the SOD and CAT activities were significantly lower (P<0.01), and the GSH-Px activity was significantly lower (P<0.05). There was no significant difference in the MDA level and SOD, GSH-Px, and CAT activities between group H and group C (P>0.05). Compared with group M, the MDA and GSH-Px levels and activities in group T were lower (P>0.05), the SOD activity was significantly higher (P<0.01), and the CAT activity was significantly higher (P<0.05). Compared with group C, the SOD activity in group T was significantly lower (P<0.01), and the other indicators did not change significantly. This shows that bovine placental polypeptides can improve intestinal oxidative stress.

[0102] 2.7 Results of relative expression of mRNA of intestinal barrier-related genes in experimental mice

[0103] like Fig.10Compared with group C, the relative expression of ZO-1 mRNA in group M was extremely significantly lower (P<0.01), the relative expression of Occludin and Claudin-1 mRNA was significantly lower (P<0.05), and the relative expression of MUC-2 mRNA was decreased (P>0.05). Compared with group C, the relative expression of ZO-1, Occludin, Claudin-1, and MUC-2 mRNA in group H was increased (P>0.05). Compared with group M, the relative expression of ZO-1 and MUC-2 mRNA in group T was significantly increased (P<0.05), and the relative expression of Occludin and Claudin-1 mRNA was increased (P>0.05), and there was no significant difference between the two groups. This indicates that bovine placental polypeptides can upregulate the expression of barrier-related genes, effectively alleviate intestinal barrier dysfunction caused by Cy, and will not have a negative impact on healthy mice.

Claims

1. Use of a cow placenta polypeptide as shown in formula I in the preparation of a drug for protecting intestinal damage caused by cyclophosphamide; 2. The use according to claim 1, characterized in that The preparation of cow placenta extract includes the following steps: Step 1, the cow placenta is cut into pieces and homogenized by a tissue homogenizer, papain is added, and the mixture is incubated at pH 6.5 and 55° C. for 4.7 h, inactivated in a boiling water bath for 10 min, and then centrifuged, the supernatant is collected, and freeze-dried to prepare a placenta enzymatic hydrolysate freeze-dried powder; The proteins and peptides in the placental enzymatic hydrolysate freeze-dried powder were identified using LC-MS / MS technology: the sample was separated using the HPLC liquid phase system Easy-nLC with a nanoliter flow rate, and then the amino acid sequence of the polypeptides in the placental enzymatic hydrolysate freeze-dried powder was analyzed by mass spectrometry using a Q Exactive mass spectrometer; Step 2: The amino acid sequence of the polypeptides in the placental hydrolysate lyophilized powder obtained in step 1 was predicted by AnOxPP (http: / / www.cqudfbp.net / AnOxPP / index.jsp), ProtParam (http: / / we-b.expasy.org / protparam / ), Innovagen (http: / / www.innovagen.com / proteomics-tools / ), ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / ) and AllerTOP (v.2.0) (https: / / www.ddgpharmfac.net / AllerTOP / ) to predict their antioxidant activity, stability, water solubility, toxicity and sensitization, and the top 5 polypeptides were screened as potential bovine placental polypeptides. Step 3: Further screening of placental peptides using molecular docking method; first, the 3D structure of potential CPAP was drawn using ChemDraw19.0 software, imported into Discovery Studio 2019 client software, and used as a ligand after removing hydrogen atoms, and TX6 (Pub Chem ID: 121488089) was selected as a positive control; secondly, the Keap1 protein crystal structure (PDB ID: 2FLU) was downloaded from the RCSB protein database (https: / / www.rcsb.org), imported into Discovery Studio 2019 client software, and used as a receptor after removing ligands and water, adding hydrogen and cleaning the protein; then, the active center (x: 5, y: 9, z: 1, radius: ), and docking was performed using the "-CDOCKER" protocol; finally, placental peptides were screened based on the -CDOCKER_INTE-RACTION_ENERGY (-CIE) score after docking; Step 4: Synthesize the placenta polypeptide by solid phase method according to its amino acid sequence, with a purity greater than 95%.

3. The use according to claim 1, characterized in that: The cow placenta polypeptide is ANNGKQWAEVF (H-Ala-Asn-Asn-Gly-Lys-Gln-Trp-Ala-Glu-Val-Phe-OH), and its molecular formula is C 57 H 82 N 16 O 17 , molecular weight is 1263.38Da.

4. The use according to claim 1, characterized in that: The cow placenta polypeptide is a drug for protecting the intestinal damage caused by cyclophosphamide.

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