Application of cow placenta peptide in preparation of product for protecting intestinal flora disorder caused by cyclophosphamide

Abstract

The invention discloses application of a dairy cow placenta peptide as shown in a formula I in preparation of a product for protecting intestinal flora disorder caused by cyclophosphamide. The dairy cow placenta peptide is QPGLPGPAG (H-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-OH), the molecular formula of the dairy cow placenta peptide is C35H56N10O11, the molecular weight of the dairy cow placenta peptide is 792.89 Da, and the dairy cow placenta peptide can be used for preparing a product for protecting intestinal flora disorder caused by cyclophosphamide. # imgabs0 #

Description

Technical Field

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

[0002] Cyclophosphamide (Cy) is widely used as a chemotherapy drug, which can lead to a decrease in the richness of intestinal bacterial populations and an increase in the number of potential pathogens, and induce the transfer of some Gram-positive bacteria to secondary lymphoid organs. Placental tissue contains a large number of bioactive substances such as peptides, cyclic peptides, steroids, collagen and amino acids. Shen Liuhong et al. and Zhang Yue et al. used different proteases to hydrolyze cow placenta and predicted the antioxidant activity of the hydrolyzate. They found that the peptides obtained after the hydrolysis of cow placenta have strong transition metal chelating ability, reducing power and ABTS free radical scavenging ability, and can inhibit the oxidative stress of aging model mice by increasing the activity of antioxidant enzymes, and delay skin aging and liver aging. However, these cow placental hydrolyzates are currently a mixture of peptides, and the active cow placental peptide components are still unclear. Therefore, the present invention uses chromatography to perform step-by-step fractionation through ion exchange, molecular sieve chromatography column and RP-HPLC, and after identifying the polypeptide sequence in the fractionated product by LC-MS / MS, selects the cow placenta peptide with the highest abundance, and applies and confirms that the cow placenta peptide can protect the intestinal flora disorder caused by cyclophosphamide. Summary of the invention

[0003] To achieve the above-mentioned purpose, the present invention provides a use of a cow placenta peptide as shown in formula I in the preparation of a product for protecting intestinal flora disorder caused by cyclophosphamide;

[0004]

[0005] To achieve the above object, the method for preparing cow placenta peptide provided by the present invention comprises the following steps:

[0006] 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 obtain cow placenta enzymatic hydrolysate freeze-dried powder;

[0007] Step 2, further separating the cow placenta hydrolysate lyophilized powder obtained in step 1 by ion exchange chromatography; taking 25mL30DEAE cellulose ion exchange resin filler and loading it into a 2×20cm glass chromatography column, and washing the equilibrated chromatographic column with 3 column volumes of phosphate buffer (pH=7.4); taking an appropriate amount of cow placenta hydrolysate lyophilized powder and adding it to phosphate buffer (pH=7.4) to prepare a 50mg / mL sample solution, and adding the sample solution 2cm away from the column surface; after loading, gradient elution was performed by phosphate buffer (pH=8.0) containing sodium chloride (0, 0.2, 0.4, 0.6mol / L), and the elution rate was 2mL / min; collecting 2mL of eluate in each tube, detecting the absorbance at 280nm, and drawing an elution curve; dividing the eluate into multiple fractions according to the elution curve; determining the DPPH free radical scavenging capacity of each fraction (the concentration of each fraction is 5mg / mL), and obtaining the fraction with the strongest antioxidant activity in step 2;

[0008] Step 3, the fraction with the strongest antioxidant activity in step 2 is separated by molecular sieve chromatography; 20g of Sephadex G25 gel filler is weighed, 200mL of deionized water is added to a boiling water bath to swell for 2h, the pH value is adjusted to 6.5, impurities suspended on the water surface are poured out, and then slowly loaded into a 2×30cm glass chromatography column, about 5cm from the upper end of the column, and 3 column volumes are balanced with deionized water at 3mL / min; the fraction with the strongest antioxidant activity obtained in step 2 is taken, deionized water is added to prepare a 20mg / mL sample solution, and then 1mL of the sample solution is taken, and the sample solution is added 2cm from the column surface, and then deionized water is used as the eluent, and the flow rate is 3mL / min for elution, and finally 2mL of the eluent is collected in each tube, the absorbance is detected at 280nm, and an elution curve is drawn; the eluent is divided into multiple fractions according to the elution curve, and the DPPH free radical scavenging ability (concentration of 5mg / mL) is measured after freeze-drying to obtain the fraction with the strongest antioxidant activity in step 3;

[0009] Step 4, using RP-HPLC to separate the fraction with the strongest antioxidant activity in step 3; using Waters 2695 high performance liquid chromatograph for separation, taking the fraction with the strongest antioxidant activity obtained in step 3; preparing the lyophilized powder of the fraction into a 5 mg / mL sample solution, using a C18 reverse phase chromatographic column (4.6x250 mm) as the analytical column, 98% ultrapure water and 2% acetonitrile as mobile phase A, 2% ultrapure water and 98% acetonitrile as mobile phase B, and using linear gradient elution after loading, with a flow rate of 0.7 mL / min, and elution conditions, 0-5 min, mobile phase B 5%-8%; 5-40 min, mobile phase B 8%-18%; 40-62 min, mobile phase B 18%-32%; 62-64 min, mobile phase B 32% to 95%; the absorbance is detected at 214 nm, and the eluate can be divided into multiple fractions according to the liquid chromatogram; after freeze-drying each fraction, the DPPH free radical scavenging ability is determined (the concentration of each fraction is 0.1 mg / mL), and the fraction with the strongest antioxidant activity in step 4 is obtained;

[0010] Step 5, using LC-MS / MS to identify the fraction with the strongest antioxidant activity in step 4; using Easy-nLC high performance liquid chromatograph connected in series with Q-Exactive mass spectrometer to identify the polypeptide amino acid sequence in the fraction with the strongest antioxidant activity obtained in step 4; dissolving the fraction in 0.1% formic acid aqueous solution as a sample solution; liquid chromatography conditions, using a C18 reverse phase chromatographic column (0.15 mm×150 mm, 3 μm) as the analytical column, 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid and 80% acetonitrile aqueous solution as mobile phase B, linear gradient elution after loading, flow rate of 600 nL / min, elution conditions, 0-2 min, mobile phase B 4%-8%; 2-45 min, mobile phase B 8%-28%; 45-55 min, mobile phase B 28%~40%; 55~56min, mobile phase B 40%~95%; mass spectrometry analysis conditions are as follows: primary mass spectrometry parameters, Resolution is 70000, AGCtarget is 3e6, MaximumIT is 100ms, Scanrange is 100to 1500m / z; secondary mass spectrometry parameters, Resolution is 17500, AGCtarget is 1e5, MaximumIT is 50ms, TopN is 20, NCE / steppedNCE is 28; finally, the cow placental peptide with the highest abundance in the identification results is selected;

[0011] Step 6: According to the amino acid sequence of the cow placenta peptide, the cow placenta peptide can be synthesized by solid phase method with a purity greater than 95%.

[0012] The cow placental peptide of the present invention is QPGLPGPAG (H-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-OH), with a molecular formula of C 35 H 56 N 10 O 11 , molecular weight 792.89Da, can be used in the preparation of products to protect intestinal flora disorders caused by cyclophosphamide.

[0013] 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.

[0014] 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

[0015] Figure 1 Chromatographic separation process and identification results.

[0016] Figure 2 This is the liquid chromatography and secondary mass spectrometry of cow placental peptides.

[0017] Figure 3 This is the OTU Venn diagram of experimental mouse fecal samples.

[0018] Figure 4 This is the dilution and Rank curve of 16S rDNA sequencing of experimental mouse feces samples.

[0019] Figure 5 Principal coordinate analysis and non-metric multidimensional scaling analysis of experimental mouse fecal samples.

[0020] Figure 6 This is a bar graph showing the bacterial flora composition at the gate level in fecal samples of experimental mice.

[0021] Figure 7 This is a bar graph showing the bacterial flora composition at the genus level in fecal samples of experimental mice.

[0022] Figure 8 This is the linear discriminant analysis score plot of experimental mouse feces samples. DETAILED DESCRIPTION

[0023] Example 1 Preparation of cow placental peptide

[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 a large-scale ranch in Sichuan; 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), pepsin (P8160), trypsin (T1300), Solarbio, USA; DPPH micro kit (R27137), Shanghai Yuanye Biotechnology Co., Ltd.; formic acid (695076), 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 hydrolysate

[0032] The cow placenta was cut into pieces and homogenized with a tissue homogenizer. Papain was added and acted at pH 6.5 and 55°C for 4.7 hours. After being inactivated in a boiling water bath for 10 minutes, the mixture was centrifuged and the supernatant was collected and freeze-dried to obtain cow placenta enzymatic hydrolysate freeze-dried powder.

[0033] 1.2.2 Isolation of cow placental peptides

[0034] First, ion exchange chromatography was used for separation: 25 mL of 30DEAE cellulose ion exchange resin filler was loaded into a 2×20 cm glass chromatography column, and 3 column volumes of phosphate buffer (pH = 7.4) were used to wash and equilibrate the chromatographic column. An appropriate amount of cow placenta enzymatic hydrolysate lyophilized powder was added to phosphate buffer (pH = 7.4) to prepare a 50 mg / mL sample solution, and the sample solution was added 2 cm from the column surface. After loading, a phosphate buffer (pH = 8.0) containing sodium chloride (0, 0.2, 0.4, 0.6 mol / L) was used for gradient elution, and the elution rate was 2 mL / min. 2 mL of eluate was collected in each tube, the absorbance was detected at 280 nm, and the elution curve was drawn. The eluate was divided into multiple fractions according to the elution curve. After desalting and freeze-drying, the DPPH free radical scavenging ability of each fraction was determined (the concentration of each fraction was 5 mg / mL), and the fraction with the strongest antioxidant activity was obtained.

[0035] Then, molecular sieve chromatography was used for separation: 20 g of Sephadex G25 gel filler was weighed, 200 mL of deionized water was added to swell in a boiling water bath for 2 h, the pH value was adjusted to 6.5, impurities suspended on the water surface were poured out, and then slowly loaded into a 2×30 cm glass chromatography column, about 5 cm from the top of the column, and 3 column volumes were balanced with deionized water at 3 mL / min. The fraction with the strongest antioxidant activity obtained in the previous step was taken, deionized water was added to prepare a 20 mg / mL sample solution, and then 1 mL of the sample solution was taken, and the sample solution was added 2 cm from the column surface, and then deionized water was used as the eluent at a flow rate of 3 mL / min for elution. Finally, 2 mL of the eluent was collected in each tube, and the absorbance was detected at 280 nm to draw an elution curve. The eluent was divided into multiple fractions according to the elution curve, and the DPPH free radical scavenging ability (concentration of 5 mg / mL) was determined after freeze-drying to obtain the fraction with the strongest antioxidant activity.

[0036] Then RP-HPLC separation was adopted: Waters 2695 high performance liquid chromatograph was used for separation, and the fraction with the strongest antioxidant activity obtained in the previous step was taken. The lyophilized powder of the fraction was prepared into a 5 mg / mL sample solution, and a C18 reverse phase chromatographic column (4.6x250 mm) was used as the analytical column, 98% ultrapure water and 2% acetonitrile were used as mobile phase A, and 2% ultrapure water and 98% acetonitrile were used as mobile phase B. After loading, linear gradient elution was adopted, and the flow rate was 0.7 mL / min. The elution conditions were as follows: 0-5 min, mobile phase B 5%-8%; 5-40 min, mobile phase B 8%-18%; 40-62 min, mobile phase B 18%-32%; 62-64 min, mobile phase B 32%-95%. The absorbance was detected at 214 nm, and the eluate could be divided into multiple fractions according to the liquid chromatogram. After freeze-drying each fraction, the DPPH free radical scavenging ability was determined (the concentration of each fraction was 0.1 mg / mL), and the fraction with the strongest antioxidant activity was obtained.

[0037] Finally, LC-MS / MS identification was performed: Easy-nLC high performance liquid chromatograph was connected to Q-Exactive mass spectrometer to identify the amino acid sequence of the polypeptide in the fraction with the strongest antioxidant activity obtained in the previous step. The fraction was dissolved in 0.1% formic acid aqueous solution as the sample solution. Liquid chromatography conditions: C18 reverse phase chromatographic column (0.15mm×150mm, 3μm) was used as the analytical column, 0.1% formic acid aqueous solution was used as mobile phase A, 0.1% formic acid and 80% acetonitrile aqueous solution was used as mobile phase B, linear gradient elution was used after loading, the flow rate was 600nL / min, elution conditions: 0-2min, mobile phase B 4%-8%; 2-45min, mobile phase B 8%-28%; 45-55min, mobile phase B 28%-40%; 55-56min, mobile phase B 40%-95%. The mass spectrometry analysis conditions are as follows: primary mass spectrometry parameters: Resolution is 70000, AGCtarget is 3e 6 , MaximumIT is 100ms, Scanrange is 100to1500m / z; Secondary mass spectrometry parameters: Resolution is 17500, AGCtarget is 1e 5 , MaximumIT is 50ms, TopN is 20, and NCE / steppedNCE is 28. The cow placenta peptide with the highest abundance in the identification results was selected, and the cow placenta peptide was synthesized by solid phase method according to the amino acid sequence of the cow placenta peptide, with a purity greater than 95%.

[0038] 1.3 Data Analysis

[0039] 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.

[0040] 2 Results and analysis

[0041] 2.1 Chromatographic separation of bovine placental peptides

[0042] In this study, ion exchange chromatography, molecular sieve chromatography and RP-HPLC were used to separate cow placental peptides from cow placental hydrolysate freeze-dried powder, and the best fraction was screened according to the DPPH free radical scavenging rate of each fraction. Finally, LC-MS / MS was used to identify the amino acid sequence of the polypeptides in the fraction. The separation and identification results are shown in Figure 2. Figure 1 The ion exchange chromatography separation results are shown in Figure 1 A, CPE was separated by 30DEAE column, and an obvious elution peak was detected at 280nm wavelength. Four fractions were obtained according to the order of elution, named D1, D2, D3, and D4. Among them, the free radical scavenging rate of D2 fraction was the highest at 5mg / mL, which was 15.20±0.24%; the free radical scavenging rate of D3 fraction was the lowest, which was 1.95±0.25% ( Figure 1 B). Therefore, fraction D2 was selected for further separation. The molecular sieve chromatography separation results are shown in Figure 1 C, using Sephadex G10 column to separate D2 fraction, a more obvious elution peak was detected at 280nm wavelength, and two fractions were obtained according to the order of peak elution, named G1 and G2. Among them, the free radical scavenging rate of G1 fraction was higher at 5mg / mL, which was 23.58±0.33%, while the free radical scavenging rate of G2 fraction was lower, which was 14.00±0.03% ( Figure 1 D). Therefore, the G1 fraction was selected for subsequent fractionation. The RP-HPLC separation results are shown in Figure 1 E. The G1 fraction was separated by high performance liquid chromatography (Waters 2695), and an obvious chromatographic peak was detected at a wavelength of 214 nm. Three fractions were obtained in the order of peak elution, named R1, R2 and R3. Among them, the free radical scavenging rate of the R2 fraction was the highest at 0.1 mg / mL, which was 10.35±0.15%, and the free radical scavenging rate of the R2 fraction was the lowest, which was 4.74±0.35% ( Figure 1F). After RP-HPLC separation, the purity of the fractionation product was the highest, so the R2 fraction was selected for subsequent peptide sequence identification. Using LC-MS / MS, combined with database retrieval software, a total of 21 peptides were identified from the R2 fraction, with the number of amino acids ranging from 7 to 11 and the molecular weight distribution ranging from 720.42 to 990.55 Da. The top 10 peptides in abundance are shown in Table 1, among which the abundance of the peptide QPGLPGPAG is much higher than that of the other peptides, and it is the main component of the R2 fraction. Therefore, the cow placental peptide QPGLPGPAG (H-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-OH) was selected, and its molecular formula is C 35 H 56 N 10 O 11 , the molecular weight is 792.89Da, and the secondary mass spectrum is as follows Figure 1 As shown in G.

[0043] Table 1 Results of partial amino acid sequence identification of R2 fraction

[0044]

[0045] 2.2 Purity analysis of synthetic cow placental peptide

[0046] Solid phase synthesis of cow placental peptide, its liquid chromatography and mass spectrum are as follows Figure 2 As shown, the cow placental peptide was found to be greater than 95% pure.

[0047] Example 2 Use of cow placental peptide in the preparation of products for protecting intestinal flora disturbance caused by cyclophosphamide

[0048] 1 Materials and methods

[0049] 1.1 Test materials

[0050] 1.1.1 Experimental animals

[0051] 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).

[0052] 1.1.2 Main reagents

[0053] Cow placental peptide QPGLPGPAG (H-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-OH), molecular formula C 35 H 56 N 10 O 11, molecular weight 792.89Da; EZNAsoil DNA kit, Omega Bio-tek, USA; AxyPrep DNA gel extraction kit, Axygen Biosciences, USA; anhydrous ethanol, Qingdao Tuobang; agarose, TE buffer (PH8.0), Beijing Bomei Fuxin.

[0054] 1.1.3 Main test instruments

[0055] Fluorometer (Qubit@2.0), bioanalyzer (Agilent), Thermo Fisher Scientific, USA; blue fluorescence quantitative system (QuantiFluorTM-ST), Beijing Promega; high-throughput sequencer (Illumina Miseq / Hiseq2500), Shanghai Zhongke.

[0056] 1.2 Test methods

[0057] 1.2.1 Grouping and dosing regimen

[0058] After 7 days of adaptive feeding, 48 mice were randomly divided into 4 groups, 12 mice per group. The control group (group C), model group (group M), and model intervention group (group T) were fed with standard diets in groups C and M, and group T was fed with standard diets + 1500 mg / kg / d cow placental peptide (according to the preliminary test results, among the four concentration gradients of 500, 1000, 1500 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 mice in group C 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.

[0059] 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.

[0060] 1.2.2 Stool sample collection and storage

[0061] Eight mice were randomly selected from each group, and rectal feces were collected and placed into sterile cryopreservation tubes. The tubes were numbered and stored in a -80°C refrigerator for the 16S rDNA detection in Experiment 2.

[0062] 1.2.3 Stool sample testing

[0063] The sequencing process is as follows: DNA extraction and detection, PCR amplification of the V3-V4 variable region of 16S rDNA using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), product purification, library preparation and quality inspection, and sequencing. The original data is obtained by sequencing, and valid data is obtained after reads splicing, tag filtering and chimera removal, and then species annotation and diversity analysis are performed. At the same time, according to the species annotation results, the species composition information at each classification level is obtained.

[0064] 1.3 Data Analysis

[0065] Based on the obtained species abundance statistics, the differences between the comparison groups were analyzed. Fisher's exact test was used to compare the differences between samples without biological replication; Mann-Whitney U test was used to compare the differences between two groups of samples with biological replication; Kruskal-Wallis test was used to compare multiple groups of samples with biological replication. Screening threshold: P < 0.05.

[0066] 2 Results and analysis

[0067] 2.1 Sequence information of experimental mouse samples

[0068] like Figure 3 After annotation, 2026, 2637, and 2170 OTUs were obtained for groups C, M, and T, respectively. There were 1001 identical OTUs between groups C and M, 1037 identical OTUs between groups C and T, 1126 identical OTUs between groups M and T, and 813 identical OTUs between groups C, M, and T.

[0069] 2.2 Sequencing quality test of experimental mice

[0070] like Figure 4 The rarefaction curve can directly reflect the rationality of the sequencing data volume. When the curve tends to be flat, it means that the sequencing data volume is reasonable, and more data will only generate a small number of new OTUs. The rarefaction curve shows a rapid rise first and then gradually flattens, indicating that the sample sequencing data volume is reasonable, the sequencing quality is good, and it has a certain depth and representativeness. As the Rank value increases, the abundance reduction trend gradually flattens, indicating that the sample species uniformity and richness are good.

[0071] 2.3 Results of bacterial diversity detection in experimental mice

[0072] As shown in Table 2 and Figure 5Compared with group C, group M had higher chao1 index, Shannon index and Simpson index (P>0.05). Compared with group M, group T had significantly lower chao1 index (P<0.05), Shannon index and Simpson index (P>0.05), and no significant difference with group C. β diversity is a comparative analysis of the composition of sample microbial communities between different samples or different groups, including principal coordinate analysis (PCoA) and non-metric multidimensional scaling analysis (NMDS), which is used to visualize and reveal the similarities and differences in sample community composition. The distance between points reflects the similarity of species composition and structure. The results showed that the samples in each group showed a very significant separation trend (P<0.01). The sample points of group C and group M were far apart, and the sample points of group T were closer to group C than those of group M. It shows that Cy can change the composition and structure of intestinal flora, and cow placental peptide can regulate the intestinal flora of mice with Cy-induced intestinal injury, making them more similar to mice in group C.

[0073] Table 2 Alpha diversity index

[0074]

[0075] 2.4 Composition and difference analysis of intestinal flora of experimental mice

[0076] After the OTUs obtained by clustering were annotated, a total of 18 phyla, 37 classes, 79 orders, 148 families, and 362 genera were obtained. The following mainly analyzes species at the phylum and genus taxonomy levels.

[0077] 2.4.1 Microbial community composition at the phylum level

[0078] like Figure 6 At the phylum level, Firmicutes, Bacteroidota, and Verrucomicrobita were the main phyla, with relative abundances in each group: 45.61%, 27.76%, and 4.15% in group C; 37.73%, 40.30%, and 10.90% in group M; and 39.59%, 36.31%, and 3.87% in group T. Compared with group C, the relative abundance of Firmicutes in group M decreased (P>0.05), the relative abundance of Patescibacteria decreased significantly (P<0.01), and the relative abundance of Bacteroidetes increased (P>0.05); compared with group M, the relative abundance of Firmicutes in group T increased (P>0.05), the relative abundance of Patescibacteria increased significantly (P<0.05), and the relative abundance of Bacteroidetes decreased (P>0.05).

[0079] 2.4.2 Bacterial composition at the genus level

[0080] like Figure 7, at the genus level, the host-associated genera Ligilactobacillus, Lactobacillus, Akkermansia, Lachnospiraceae_NK4A136_group, Desulfovibrio, Candidatus_Saccharimonas, Carnobacterium, Alistipes, Psychrobacter, and Dubosiella were the most dominant bacterial groups, among which the host-associated Lactobacillus had the highest abundance (Group C: 10.02%, Group M: 4.80%, Group T: 7.84%). In addition, a differential test was performed on the top 20 genera with relative species abundances. The abundances of Candidatus_Saccharimonas and Enterobacteriaceae were extremely significantly decreased in Group M compared with Group C (P<0.01), the abundances of Carnobacterium and Psychrobacter showed a decreasing trend (0.05<P<0.1), the abundance of Dubosiella was significantly increased (P<0.05), and the abundance of Allobaculum was extremely significantly increased (P<0.01). The abundances of Candidatus_Saccharimonas and Psychrobacter were significantly increased in Group T compared with Group M (P<0.05), the abundance of Carnobacterium was extremely significantly decreased (P<0.01), and the abundance of Allobaculum showed a decreasing trend (0.05<P<0.1).

[0081] 2.5 Differences in the bacterial community structure

[0082] As Figure 8 , the differences in species were analyzed by linear discriminant analysis, and the effects of the abundances of bacterial community species in each group on the difference were compared by combining the linear discriminant analysis effect sizes (LEfSe) and P value to screen out the differential species (LDA score ≥ 3, P<0.05). The number of bacterial groups in Group C was more than that in Group M, and the number of groups in Group T was the least. The main bacterial groups in Group C were Carnobacterium, Candidatus_Saccharimonas, Psychrobacter, Acinetobacter, Enterobacteriaceae, Streptococcus, Monococcus, Aerococcus, and Pantoea. The main bacterial groups in Group M were Dubosiella, Psychrobacillus, Allobaculum, Turicibacter, and Coprobacillus. The main bacterial groups in Group T were Rikenellaceae_RC9_gut_group and Enterobacteriaceae.

Claims

1. Use of a cow placental peptide as shown in formula I in the preparation of a product for protecting intestinal flora disorder caused by cyclophosphamide; 2. The use according to claim 1, characterized in that The preparation of cow placenta peptide 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 obtain cow placenta enzymatic hydrolysate freeze-dried powder; Step 2, further separating the cow placenta hydrolysate lyophilized powder obtained in step 1 by ion exchange chromatography; taking 25 mL of 30DEAE cellulose ion exchange resin filler and loading it into a 2×20 cm glass chromatography column, and washing and balancing the chromatographic column with 3 column volumes of phosphate buffer (pH=7.4); taking an appropriate amount of cow placenta hydrolysate lyophilized powder and adding it to phosphate buffer (pH=7.4) to prepare a 50 mg / mL sample solution, and adding the sample solution 2 cm away from the column surface; After loading, phosphate buffer (pH=8.0) containing sodium chloride (0, 0.2, 0.4, 0.6 mol / L) was used for gradient elution at a rate of 2 mL / min; 2 mL of the eluate was collected in each tube, the absorbance was detected at 280 nm, and the elution curve was drawn; The eluate was divided into multiple fractions according to the elution curve; the DPPH free radical scavenging capacity of each fraction was measured (the concentration of each fraction was 5 mg / mL), and the fraction with the strongest antioxidant activity in step 2 was obtained; Step 3, the fraction with the strongest antioxidant activity in step 2 is separated by molecular sieve chromatography; 20 g of Sephadex G25 gel filler is weighed, 200 mL of deionized water is added to swell in a boiling water bath for 2 h, the pH value is adjusted to 6.5, impurities suspended on the water surface are poured out, and then slowly loaded into a 2×30 cm glass chromatography column, about 5 cm from the top of the column, and 3 column volumes are balanced with deionized water at 3 mL / min; the fraction with the strongest antioxidant activity obtained in step 2 is taken, deionized water is added to prepare a 20 mg / mL sample solution, and then 1 mL of the sample solution is taken, and the sample solution is added 2 cm from the column surface, and then deionized water is used as the eluent at a flow rate of 3 mL / min for elution, and finally 2 mL of the eluent is collected in each tube, the absorbance is detected at 280 nm, and an elution curve is drawn; The eluate was divided into multiple fractions according to the elution curve, and the DPPH free radical scavenging ability was measured after freeze-drying (concentration was 5 mg / mL), and the fraction with the strongest antioxidant activity in step 3 was obtained; Step 4, using RP-HPLC to separate the fraction with the strongest antioxidant activity in step 3; using Waters 2695 high performance liquid chromatograph for separation, taking the fraction with the strongest antioxidant activity obtained in step 3; preparing the lyophilized powder of the fraction into a 5 mg / mL sample solution, using a C18 reverse phase chromatographic column (4.6x250 mm) as the analytical column, 98% ultrapure water and 2% acetonitrile as mobile phase A, 2% ultrapure water and 98% acetonitrile as mobile phase B, and using linear gradient elution after loading, with a flow rate of 0.7 mL / min, and elution conditions, 0-5 min, mobile phase B 5%-8%; 5-40 min, mobile phase B 8% to 18%; 40 to 62 min, mobile phase B 18% to 32%; 62 to 64 min, mobile phase B 32% to 95%; detect absorbance at 214 nm, and the eluate can be divided into multiple fractions according to the liquid chromatogram; after freeze-drying each fraction, measure the DPPH free radical scavenging ability (the concentration of each fraction is 0.1 mg / mL), and obtain the fraction with the strongest antioxidant activity in step 4; Step 5, using LC-MS / MS to identify the fraction with the strongest antioxidant activity in step 4; using Easy-nLC high performance liquid chromatograph connected in series with Q-Exactive mass spectrometer to identify the polypeptide amino acid sequence in the fraction with the strongest antioxidant activity obtained in step 4; dissolving the fraction in 0.1% formic acid aqueous solution as a sample solution; liquid chromatography conditions, using a C18 reverse phase chromatographic column (0.15 mm×150 mm, 3 μm) as the analytical column, 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid and 80% acetonitrile aqueous solution as mobile phase B, linear gradient elution after loading, flow rate of 600 nL / min, elution conditions, 0-2 min, mobile phase B 4%-8%; 2-45 min, mobile phase B 8%-28%; 45-55 min, mobile phase B 28%-40%; 55-56 min, mobile phase B 40%~95%; the mass spectrometry analysis conditions are as follows: primary mass spectrometry parameters, Resolution is 70000, AGCtarget is 3e6, MaximumIT is 100ms, Scanrange is 100to 1500m / z; secondary mass spectrometry parameters, Resolution is 17500, AGCtarget is 1e5, MaximumIT is 50ms, TopN is 20, NCE / steppedNCE is 28; finally, the cow placental peptide with the highest abundance in the identification results is selected; Step 6: According to the amino acid sequence of the cow placenta peptide, it can be synthesized by solid phase method with a purity greater than 95%.

3. The use according to claim 1, characterized in that: The cow placental peptide is QPGLPGPAG (H-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Ala-Gly-OH), with a molecular formula of C 35 H 56 N 10 O 11 , molecular weight 792.89Da.

4. The use according to claim 1, characterized in that: The cow placenta peptide can be used in the preparation of a product for protecting the intestinal flora disorder caused by cyclophosphamide.

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