Nonapeptide with function of promoting peristalsis as well as preparation method and application of nonapeptide

Abstract

The invention discloses nonapeptide with an effect of promoting peristalsis as well as a preparation method and application of the nonapeptide, and belongs to the technical field of biological medicines. The amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln, and the amino acid sequence of the nonapeptide PQ9 is as follows: Pro-Gly-Tyr-Gln. The peptide fragment can be obtained through artificial synthesis or directional enzymolysis of wheat peptide. Functional verification shows that the polypeptide has the function of promoting peristalsis, and the function is mainly reflected in shortening the time interval of peristalsis peaks and improving the peristalsis promoting rate. The invention provides a novel active substance for promoting peristalsis, which can be applied to preparation of health food or medicine for improving peristalsis disorder, and has good market prospect and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a nonapeptide with the function of promoting intestinal peristalsis, and a preparation method and application thereof. Background Art

[0002] Intestinal peristalsis refers to the movement similar to that of worms, especially the wave-like contraction of the intestine. Small intestine peristalsis occurs when food, which has been preliminarily digested in the stomach, enters the small intestine, and the small intestine produces peristalsis when absorbing nutrients; large intestine peristalsis occurs when the food is digested and enters the large intestine waiting to be excreted from the body. Intestinal peristalsis dysfunction means that the peristalsis of the intestine is not strong enough or too slow, resulting in the dysfunction of the digestive system. The slowdown of intestinal peristalsis cannot ensure the rapid excretion of toxins in the body, leading to the continuous accumulation of harmful substances in the body. Long-term slowdown of peristalsis will cause gastrointestinal diseases such as intestinal obstruction and constipation, and can also trigger cardiovascular and cerebrovascular diseases.

[0003] Due to being affected by various factors, the exact pathogenesis of peristalsis dysfunction has not been clarified yet. At present, laxatives are mostly relied on to improve this symptom. However, laxatives have different degrees of side effects on the human body. For example, long-term use of anthraquinone laxatives can lead to melanosis coli and has the risk of suffering from colorectal cancer. Therefore, it is particularly important to find safe and effective natural substances to promote intestinal peristalsis.

[0004] Food-derived bioactive peptides can maintain the intestinal barrier function by regulating the proliferation and composition of intestinal epithelial cells, reducing oxidative stress of intestinal cells, stimulating the production and secretion of mucins and antimicrobial peptides into the lumen, maintaining the composition and function of endocrine cells, supporting the expression and cell distribution of tight junction proteins, regulating the diversity and metabolism of symbiotic microorganisms, comprehensively enhancing the function of the intestinal mucosa, and affecting the inflammatory state of intestinal cells and the activity of underlying lamina propria immune cells. Therefore, food-derived bioactive peptides can actively affect gastrointestinal homeostasis by regulating barrier function, immune response and intestinal microbiota.

[0005] For example, Zhang Ting et al. reported that walnut oligopeptides have the function of moistening the intestine and relieving constipation by increasing the small intestine propulsion rate, shortening the time for the first black stool to be excreted, and regulating the levels of gastrointestinal hormones. The walnut oligopeptides are extracted from walnuts by using a biological enzymolysis technology, and the main component is small molecule oligopeptides with a relative molecular weight of <1000 Da and a content of >95% (Function of walnut oligopeptides in moistening the intestine and relieving constipation. Chinese Journal of Public Health, 2019, 35(9): 1225-1228.); Patent document CN114588247A discloses an active peptide probiotic composition containing bovine liver peptides (<1 kDa) and soybean peptides (<1 kDa), which has the functions of promoting intestinal peristalsis and relieving constipation; Patent document CN112226478A discloses that wheat peptides (prepared from wheat gluten by biological enzymolysis, refining and spray drying) have the function of promoting and relieving constipation.

[0006] The above research objects are all mixtures of bioactive peptides. At present, the research on small molecule peptides in this field that can promote intestinal peristalsis is insufficient. Therefore, it is necessary to analyze the peptide spectrum of protease hydrolysis products to screen out bioactive small molecule peptides related to the function of promoting intestinal peristalsis. Summary of the Invention

[0007] The purpose of the present invention is to provide a natural small molecule bioactive peptide with the function of promoting intestinal peristalsis, and apply it to the development of drugs or health foods for improving intestinal peristalsis dysfunction.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions: The present invention uses LC-MS / MS peptide spectrum analysis technology to analyze the polypeptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between peptide segments and acetylcholine receptors. A candidate peptide segment is screened out. After mass spectrometry identification, its amino acid sequence is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln (PGYQVHWPQ), and its molecular weight is 1110.52 Da. It is named PQ9. Further, the nonapeptide PQ9 is synthesized artificially, and functional verification shows that the nonapeptide PQ9 exhibits a significant effect of promoting intestinal peristalsis in an animal model of intestinal peristalsis disorder.

[0009] Therefore, the present invention provides a bioactive nonapeptide PQ9, and the amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

[0010] The present invention also provides a method for preparing the nonapeptide PQ9. The nonapeptide PQ9 can be prepared by solid-phase synthesis method. The specific method includes: adopting Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as the solid-phase carrier, and introducing glutamine, proline, tryptophan, histidine, valine, glutamine, tyrosine, glycine, and proline residues in sequence to extend the peptide chain from the C-terminus to the N-terminus, and solid-phase synthesize the nonapeptide PQ9.

[0011] The nonapeptide PQ9 can also be obtained by enzymatic hydrolysis of wheat gluten. Specifically, wheat protein (gluten powder) and water are mixed at a mass ratio of 1:15 - 20, the pH of the material-liquid is adjusted to 8.0 ± 0.2, and alkaline protease accounting for 0.5% of the total weight of the gluten powder is added for enzymatic hydrolysis for 30 min, and shearing is carried out for 15 - 30 min under the condition of a rotation speed of 10,000 - 15,000 r / min; after the shearing is completed, the pH of the material-liquid is adjusted to 8.0 ± 0.2, and then alkaline protease accounting for 1.5% of the total weight of the gluten powder is added in sequence for continuous enzymatic hydrolysis for 30 min, neutral protease accounting for 0.5% of the total weight of the gluten powder is added for continuous enzymatic hydrolysis for 45 min, and flavor protease accounting for 0.5% of the total weight of the gluten powder is added for 30 min; after the enzymatic hydrolysis is completed, centrifugation is carried out under the condition of a rotation speed of 6,000 r / min, and the supernatant is taken and filtered through a membrane with a molecular weight cut-off of 5 kDa, and the obtained filtrate contains the nonapeptide PQ9.

[0012] The present invention also provides the application of the nonapeptide PQ9 in the preparation of drugs or health foods for improving intestinal peristalsis disorders and / or relieving constipation. The research of the present invention shows that the nonapeptide PQ9 has the effect of promoting intestinal peristalsis. Administering PQ9 treatment to the animal model of intestinal peristalsis disorder can significantly promote intestinal peristalsis and improve the condition of intestinal peristalsis disorder, and it can be applied to the development of related products for improving intestinal peristalsis disorders.

[0013] Furthermore, the promotion of intestinal peristalsis includes at least one of shortening the time interval between intestinal peristalsis peaks and increasing the intestinal peristalsis promotion rate.

[0014] The present invention provides a pharmaceutical composition for treating diseases related to intestinal peristalsis disorders. The active ingredient of the pharmaceutical composition includes the nonapeptide PQ9, and the amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln. The diseases related to intestinal peristalsis disorders can be, but are not limited to, intestinal obstruction and constipation.

[0015] In the pharmaceutical composition provided by the present invention, the nonapeptide PQ9 can be used as the only active ingredient for exerting the effect of promoting intestinal peristalsis, or can be used in combination with other active ingredients having the function of promoting intestinal peristalsis. The active components having the function of promoting intestinal peristalsis can be, but are not limited to, dietary fiber, probiotics or other protein peptides.

[0016] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier. The present invention takes the nonapeptide PQ9 as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a preparation according to the preparation method of the preparation recorded in pharmacy.

[0017] Furthermore, the pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder and a lubricant.

[0018] Furthermore, the pharmaceutical composition can be in a dosage form of, but not limited to, oral preparations. Specifically, the dosage form can be, but not limited to, oral liquids, capsules, microcapsule powders, tablets, granules or emulsions.

[0019] The present invention also provides a health food for promoting intestinal peristalsis, which comprises nonapeptide PQ9 as an active ingredient and a food pharmaceutically acceptable excipient. The amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln. The function of the health food is to promote digestion or defecation.

[0020] Furthermore, the dosage form of the health food is a beverage, an oral liquid, a capsule, a microcapsule powder, a tablet, a granule or an emulsion.

[0021] The beneficial effects of the present invention are as follows: The present invention provides a nonapeptide PGYQVHWPQ with the function of promoting intestinal peristalsis. This peptide segment can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat peptides. Functional verification in animal models shows that this nonapeptide has the function of promoting intestinal peristalsis, which is mainly reflected in shortening the time interval between intestinal peristalsis peaks and increasing the promotion rate of intestinal peristalsis. Moreover, the nonapeptide PGYQVHWPQ is a natural peptide segment with high biological safety. Therefore, it can be applied to the preparation of health foods or drugs for improving intestinal peristalsis disorders. The present invention provides a new solution for improving intestinal peristalsis disorders and has good market prospects and application potential. Description of the Drawings

[0022] Figure 1 It is the secondary mass spectrometry diagram of nonapeptide PQ9. In the figure, #1:1 represents the number; P / G / Y / Q / V / H / W / P / Q represents the nonapeptide sequence; the vertical coordinate Intensity(%) represents the relative abundance; b2 represents the second fragment ion generated by the cleavage at the N-terminus of the peptide segment; y2 represents the second fragment ion generated by the cleavage at the C-terminus of the peptide segment; y3 represents the third fragment ion generated by the cleavage at the C-terminus of the peptide segment; b4 represents the fourth fragment ion generated by the cleavage at the N-terminus of the peptide segment; b5 represents the fifth fragment ion generated by the cleavage at the N-terminus of the peptide segment; y4 represents the fourth fragment ion generated by the cleavage at the C-terminus of the peptide segment; y5 represents the fifth fragment ion generated by the cleavage at the C-terminus of the peptide segment; b6 represents the sixth fragment ion generated by the cleavage at the N-terminus of the peptide segment; y6 represents the sixth fragment ion generated by the cleavage at the C-terminus of the peptide segment; b7 represents the seventh fragment ion generated by the cleavage at the N-terminus of the peptide segment; y7 represents the seventh fragment ion generated by the cleavage at the C-terminus of the peptide segment; b8 represents the eighth fragment ion generated by the cleavage at the N-terminus of the peptide segment; y8 represents the eighth fragment ion generated by the cleavage at the C-terminus of the peptide segment.

[0023] Figure 2Schematic diagram of the binding of nonapeptide PQ9 to CHRM1.

[0024] Figure 3 Intestinal peristalsis pictures recorded by a stereomicroscope after nonapeptide PQ9 and LH9 act on the zebrafish model for comparison.

[0025] Figure 4 To compare the effects of nonapeptide PQ9 and LH9 on the time interval of zebrafish intestinal peristalsis, where the * symbol indicates a significant difference compared with the blank group (NC), * indicates P <0.05; the # symbol indicates a significant difference compared with the constipation model group (MC), ## indicates P <0.01.

[0026] Figure 5 Pictures under a stereofluorescence microscope after nonapeptide PQ9 and LH9 act on the zebrafish model for comparison.

[0027] Figure 6 To compare the effects of nonapeptide PQ9 and LH9 on the promotion rate of zebrafish intestinal peristalsis, where the * symbol indicates a significant difference compared with the blank group (NC), * indicates P <0.05, ** indicates P <0.01.

[0028] Figure 7 Intestinal peristalsis pictures recorded by a stereomicroscope after nonapeptide PQ9 and wheat peptide act on the zebrafish model for comparison.

[0029] Figure 8 To compare the effects of nonapeptide PQ9 and wheat peptide on the time interval of zebrafish intestinal peristalsis, where the * symbol indicates a significant difference compared with the blank group (NC), * indicates P <0.05; the # symbol indicates a significant difference compared with the constipation model group (MC), ## indicates P <0.01, indicates P <0.001.

[0030] Figure 9 Pictures under a stereofluorescence microscope after nonapeptide PQ9 and wheat peptide act on the zebrafish model for comparison.

[0031] Figure 10 To compare the effects of nonapeptide PQ9 and wheat peptide on the promotion rate of zebrafish intestinal peristalsis, where the * symbol indicates a significant difference compared with the blank group (NC), *** indicates P <0.001. Specific implementation mode

[0032] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0033] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0034] Wheat protein (gluten) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.

[0035] Example 1: Screening of active peptide segments 1. Preparation of wheat peptide (glutamine peptide) According to the ratio of wheat protein (gluten) to water of 1:15, it was put into an enzymatic hydrolysis reaction tank, the pH of the feed liquid was adjusted to 8.0 ± 0.2, and 0.5% of alkaline protease (derived from Bacillus licheniformis) based on the total weight of gluten was added for enzymatic hydrolysis for 30 min. Then, a shearing machine was started, and the shearing speed was 15,000 r / min for 30 min.

[0036] After the shearing was completed, the pH of the feed liquid was adjusted to 8.0 ± 0.2, and 1.5% of alkaline protease (derived from Bacillus licheniformis) based on the total weight of gluten was added to continue enzymatic hydrolysis for 30 min. After completion, 0.5% of neutral protease (derived from Bacillus subtilis) based on the total weight of gluten was added to continue enzymatic hydrolysis for 45 min. After completion, 0.5% of flavor protease was added and reacted for 30 min.

[0037] After the enzymatic hydrolysis was completed, separation was carried out by a disc centrifuge (rotating speed 6000 r / min), the supernatant was taken and filtered through a membrane with a molecular weight cut-off of 5 kDa. After filtration, the supernatant was taken, sterilized and spray-dried to obtain wheat peptide (glutamine peptide) powder.

[0038] 2. Screening of active peptide segments LC-MS / MS peptide spectrum analysis was carried out on wheat peptides, and the obtained peptide segments of wheat peptides were sorted according to the screening conditions. Subsequently, molecular docking was further used to determine the final theoretical effective peptide segments according to the scores. The specific analysis process is as follows: (1) Identification of wheat peptide sequence Dissolve the wheat peptide sample in NH4HCO3 solution, add dithiothreitol solution, and place it in a water bath at 56 °C for 1 h for reduction. Subsequently, add iodoacetamide solution and react in the dark for 40 min. After desalting, evaporate the solvent to dryness, and then dissolve the sample in 10 μL of mobile phase A (0.1% formic acid) into a liquid-phase injection vial. Subsequently, LC-MS / MS analysis is carried out.

[0039] Chromatographic conditions: Analytical column (Acclaim PepMap RPLC C18, 150×150 mm, 3 μm); Mobile phase A (0.1% formic acid); Mobile phase B (0.1% formic acid and 80% acetonitrile); Flow rate (600 nL / min). Gradient elution program: 0 - 2 min, 4% B - 8% B; 2 - 45 min, 8% B - 40% B; 45 - 55 min, 40% B - 60% B; 55 - 56 min, 60% B - 95% B; 56 - 66 min, 95% B.

[0040] Mass spectrometry conditions: Full-scan MS uses Orbitrap for primary scanning, scanning range (100~1500 m / z), resolution (70,000), maximum ion injection time (100 ms), automatic gain control (3×106); Use high-energy collision dissociation to fragment the top 20 precursor ions that meet the tandem (MS / MS) fragmentation conditions and scan with Orbitrap, resolution (17,500), maximum ion injection time (50 ms), automatic gain control (1×10 5 ). The raw data obtained by mass spectrometry is analyzed using the De novo software of PEAKSStudio for polypeptide sequence analysis.

[0041] (2) Screening of active peptides with potential function of promoting intestinal peristalsis The enteric nervous system is the largest component of the peripheral nervous system and can independently control intestinal functions without the central nervous system. Severe constipation patients, especially those who have long-term taken stimulant laxatives, often show enteric nervous system dysfunctions including a decrease in the number of colonic neurons and damage to cell populations. In intestinal neurons, the activation of muscarinic acetylcholine receptor (mAChR) can lead to calcium ion influx and trigger a series of intracellular signal transduction, thus participating in the regulation of intestinal motility, secretion, and other physiological processes. CHRM1 is one of the mAChR subtypes. Previous studies found that wheat peptides (mixture) can effectively promote intestinal peristalsis and activate CHRM1. Therefore, molecular docking of wheat peptide fragments with CHRM1 is performed to screen small peptide fragments that may have the function of promoting intestinal peristalsis.

[0042] Specifically, according to the average local confidence (ALC) greater than 95% and the peak area greater than 2×10 6, Peptide segments that meet the criteria were screened under the condition that the PeptideRanker score is greater than 0.8. Subsequently, the peptide segments were subjected to molecular docking with the M1-muscarinic acetylcholine receptor (CHRM1).

[0043] First, the crystal structure (5CXV) of CHRM1 was downloaded from the PDB protein database. After removing water molecules and adding hydrogen atoms to the receptor target through Discovery Studio software, its active center was defined. The structure of the selected wheat peptide segments was constructed by Discovery Studio, and its energy was minimized by the CHARMm force field. These peptides were defined as ligands. The constructed peptides were docked with CHRM1 by CDOCKER to simulate the binding mode, site, and amino acid residues that interact with the lowest binding energy and the highest binding degree, and were screened according to the binding energy and the number of hydrogen bonds. Finally, two nonapeptides (PGYQVHWPQ and LNPFPFEEH) were determined, as shown in Table 1.

[0044] Table 1. Peptide segments with potential intestinal peristalsis promoting activity in wheat peptides Peptide sequence ALC score (%) Length Mass-to-charge ratio (m / z) Charge number (z) Relative abundance Molecular weight (Da) Peptide Ranker score Docking energy (kcal / mol) PGYQVHWPQ 95.8 9 556.268 2 5.25E+06 1110.52 0.51 -101.45 LNPFPFEEH 95 9 565.76 2 3.36E+06 1129.51 0.53 -114.11

[0045] Among them, the secondary mass spectrum of the nonapeptide PQ9 is as Figure 1 shown. The cleavage fragment ions of peptides include: N-terminal fragment ions (types a, b, c) and C-terminal fragment ions (types x, y, z). The side chain cleavage of a, y, and z type ions forms d, v, w type ions respectively. In addition, there are internal ions formed by the cleavage at both ends, etc. The b and y series ions are the most common. The primary structure of the peptide can be deduced based on the b or y series fragment ions of the peptide. 556.268 m / z is the [M+H] + ion signal of the nonapeptide, the charge number (z) is 2, and the molecular weight is 1110.52 Da, which is basically consistent with the nonapeptide PGYQVHWPQ. Further, the nonapeptide was analyzed by in-source collision-induced dissociation technology for secondary mass spectrometry (as Figure 1 shown), and the primary structure of the nonapeptide was determined to be Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

[0046] The 2D and 3D diagrams of the molecular docking of PQ9 with CHRM1 are as Figure 2As shown, the analysis of chemical bonds reveals that PQ9 binds to CHRM1 mainly through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and hydrophobic interactions (π-alkyl), with a docking energy of -101.45 kcal / mol. PQ9 forms 14 van der Waals forces with amino acid residues PRO1142, ASN1143, GLN1140, ARG1147, LEU1031, GLY1106, GLU1107, TRP1137, THR1141, THR1025, ASP1019, LYS1018, GLY1011, and ARG1013, 16 hydrogen bonds with ASP1009, THR1020, GLY1029, PHE1103, GLU1021, GLN1104, GLU1010, ARG1144, VAL1102, and MET1105, and 1 hydrophobic interaction with ARG1144.

[0047] From the molecular docking results, it can be seen that at the molecular docking level, the nonapeptide PQ9 can bind to CHRM1, and then activate CHRM1 to play a role in regulating intestinal motility.

[0048] The peptide segments PGYQVHWPQ and LNPFPFEEH were synthesized by Shenzhen Borun Sida Biotechnology Co., Ltd. with a purity of ≥98% for subsequent functional verification.

[0049] Example 2: Comparison of the effects of nonapeptide PQ9 and LH9 on intestinal peristalsis in a zebrafish intestinal peristalsis disorder model 1. Comparison of the effects of nonapeptide PQ9 and LH9 on the time interval of intestinal peristalsis in zebrafish In this example, wild-type AB strain zebrafish were used for the study. By measuring the time interval between two intestinal peristalsis wave peaks in zebrafish, the prokinetic effect of the sample was evaluated.

[0050] AB-type zebrafish (4 months old) were raised in a circulating water system at 28 ± 0.5 °C and pH 7.0 - 7.5, with a photoperiod of 14 h light / 10 h dark, and were fed Artemia twice a day at a fixed time. Healthy adult zebrafish were placed in a special mating tank at a ratio of 1:1 of male to female the evening before, with the water level about 2 / 3, and the male and female fish were separated by a partition. At about 8 am the next day, the partition was removed to allow the zebrafish to fertilize freely, and zebrafish embryos were obtained. At this time, the embryos were 0 hpf. After removing the impurities, the embryos were placed in a petri dish containing methylene blue system water, and the petri dish was placed in an incubator at a constant temperature of 28 ± 0.5 °C. After 24 h, phenylthiourea (PTU) was added to inhibit melanin synthesis.

[0051] Zebrafish at 5 dpf were selected and placed in six-well plates, divided into 4 groups, with 20 fish in each well. The specific grouping was the blank group (NC), the model group (MC), the PQ9 group (5 μg / mL), and the LH9 group (5 μg / mL). The blank group was added with the same volume of system water, the model group was added with 10 g / mL ropivacaine hydrochloride, and the sample groups were added with the corresponding doses of samples and ropivacaine hydrochloride at the same time. After culturing in a constant temperature incubator at 28 ± 0.5°C for 24 h, the intestinal peristalsis videos of zebrafish in each group were recorded with a stereomicroscope. After the experiment, by repeatedly watching the videos, the time interval between two intestinal peristalsis peaks of zebrafish was recorded.

[0052] In this study, Graphpad Prism 8.0 software was used for statistical difference analysis and drawing, and all data were expressed as mean ± standard error. One-way analysis of variance was used to compare the differences between groups (* P < 0.05, ** P < 0.01, *** P < 0.001; compared with the NC group, # P < 0.05, ## P < 0.01, P < 0.001).

[0053] The results are as Figure 3 and Figure 4 shown. Compared with the NC group, the intestinal peristalsis time interval in the MC group increased significantly ( P < 0.05), indicating that the intestinal peristalsis disorder model was successfully established. Compared with the MC group, although LH9 treatment shortened the intestinal peristalsis time interval, it did not reach significance ( P > 0.05), while PQ9 treatment could significantly shorten the intestinal peristalsis time interval ( P < 0.01), indicating that PQ9 has a significant effect on the intestinal peristalsis time interval.

[0054] 2. Compare the effects of the nonapeptide PQ9 and LH9 on the intestinal peristalsis promotion rate of zebrafish The fluorescent dye Nile Red is not interfered by other tissues in zebrafish and is not absorbed. The promotion effect of intestinal peristalsis can be evaluated by quantifying Nile Red in the gastrointestinal tract of zebrafish.

[0055] Zebrafish at 5 dpf were stained with Nile red dye and cultured in the dark for 16 h. Subsequently, the dye was washed off with systematic water, and the zebrafish were transferred to a 6-well plate and divided into 3 groups, with 20 fish in each well. The specific grouping was the blank group, the PQ9 group (5 μg / mL), and the LH9 group (5 μg / mL). The blank group was added with the same volume of systematic water, and the sample groups were added with the corresponding doses of the samples. After culturing in the dark for 24 h, the zebrafish were photographed under a stereomicroscope with the same magnification, fluorescence intensity, exposure time, and fluorescence gain. The total fluorescence signal in the zebrafish intestine was analyzed using the NIS-Elements D 3.10 advanced image processing software, and the promoting effect of the samples on zebrafish intestinal peristalsis was judged by the amplitude of the decrease in fluorescence intensity before and after. The calculation method was as follows: Promotion rate of intestinal peristalsis = (Sblank control group - Ssample group) / Sblank control group × 100% The results were as Figure 5 and Figure 6 shown. Compared with the NC group, both the nonapeptides PQ9 and LH9 could significantly increase the promotion rate of intestinal peristalsis ( P < 0.01 and P < 0.05), indicating that both PQ9 and LH9 had significant effects on the promotion rate of intestinal peristalsis, but the effect of LH9 was lower than that of PQ9.

[0056] Based on the results of the intestinal peristalsis peak time interval and the promotion rate of intestinal peristalsis, it can be seen that the nonapeptide PQ9 has the effect of promoting intestinal peristalsis.

[0057] Example 3: Comparison of the effects of nonapeptide PQ9 and wheat peptide on intestinal peristalsis in a zebrafish intestinal peristalsis disorder model 1. Comparison of the effects of nonapeptide PQ9 and wheat peptide on the intestinal peristalsis time interval in zebrafish The test method was the same as that in "Comparison of the effects of nonapeptide PQ9 and LH9 on the intestinal peristalsis time interval in zebrafish". The grouping was as follows: divided into 8 groups, with 20 fish in each well, including the blank group (NC), the model group (MC), the low-dose wheat peptide group (10 μg / mL, WP-10) and the high-dose wheat peptide group (100 μg / mL, WP-100), the lowest-dose PQ9 group (0.5 μg / mL, PQ9-0.5), the low-dose PQ9 group (1 μg / mL, PQ9-1), the medium-dose PQ9 group (5 μg / mL, PQ9-5), and the high-dose PQ9 group (10 μg / mL, PQ9-10).

[0058] The results were as Figure 7 and Figure 8 shown. Compared with the NC group, the intestinal peristalsis time interval in the MC group increased significantly ( P < 0.05), indicating that the intestinal peristalsis disorder model was successfully established. Compared with the MC group, although the wheat peptide at 10 μg / mL shortened the intestinal peristalsis time interval, it did not reach a significant level ( P> 0.05), when the concentration of wheat peptide is 100 μg / mL, it can significantly shorten the intestinal peristalsis time interval ( P < 0.01). When the concentration of nonapeptide PQ9 is 5 μg / mL and 10 μg / mL, it can significantly shorten the intestinal peristalsis time interval ( P < 0.001). Based on the above data, it can be known that from the perspective of the intestinal peristalsis time interval, the promoting effect of nonapeptide PQ9 on intestinal peristalsis is about 20 times that of wheat peptide.

[0059] 2. Compare the effects of nonapeptide PQ9 and wheat peptide on the promotion rate of zebrafish intestinal peristalsis The test method is the same as that of "Compare the effects of nonapeptide PQ9 and LH9 on the promotion rate of zebrafish intestinal peristalsis". The grouping is as follows: divided into 7 groups, 20 tails per well, including the blank group, low-dose (10 μg / mL) and high-dose (100 μg / mL) wheat peptide groups, lower-dose (0.5 μg / mL), low-dose (1 μg / mL), medium-dose (5 μg / mL) and high-dose (10 μg / mL) PQ9 groups.

[0060] The results are as Figure 9 and Figure 10 shown. Compared with the NC group, although 10 μg / mL of wheat peptide increased the promotion rate of intestinal peristalsis, it did not reach significance ( P > 0.05), when the concentration of wheat peptide is 100 μg / mL, it can significantly increase the promotion rate of intestinal peristalsis ( P < 0.001). When the concentration of nonapeptide PQ9 is 1 - 10 μg / mL, it can significantly increase the promotion rate of intestinal peristalsis ( P < 0.001). Based on the above data, it can be known that from the perspective of the promotion rate of intestinal peristalsis, the promoting effect of nonapeptide PQ9 on intestinal peristalsis is about 100 times that of wheat peptide.

[0061] In summary, through mass spectrometry identification and molecular docking of peptide segments, the present invention screened nonapeptide PGYQVHWPQ from wheat peptides. This peptide segment shows high activity in promoting intestinal peristalsis in the intestinal peristalsis disorder model, mainly reflected in shortening the peak time interval of intestinal peristalsis and increasing the promotion rate of intestinal peristalsis. The present invention provides a theoretical basis for the development of new active substances for promoting intestinal peristalsis, which can be applied to health foods or drugs for improving intestinal peristalsis disorders. Nonapeptide PGYQVHWPQ can be used alone to prepare health foods or drugs for improving intestinal peristalsis disorders, or can be compounded with other active ingredients with the function of promoting intestinal peristalsis. The active components with the function of promoting intestinal peristalsis can be, but are not limited to, dietary fiber, probiotics or other protein peptides. The health food or drug can be prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability and shelf life of the polypeptide, so that it can be better applied to the food industry and health care field.

Claims

1. A bioactive nonapeptide PQ9, characterized in that, The amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

2. The preparation method of the nonapeptide PQ9 according to claim 1, characterized in that, The nonapeptide PQ9 is prepared by solid-phase synthesis; or obtained by enzymatic hydrolysis of wheat gluten.

3. Use of the nonapeptide PQ9 as claimed in claim 1 in the preparation of a medicament or health food for improving intestinal motility disorders and / or relieving constipation, characterized in that, The nonapeptide PQ9 has the effect of promoting intestinal peristalsis.

4. The application according to claim 3, characterized in that The promotion of intestinal peristalsis includes at least one of shortening the time interval between intestinal peristalsis peaks and increasing the intestinal peristalsis promotion rate.

5. A pharmaceutical composition for treating diseases related to intestinal motility disorders, characterized in that, The active ingredient of the pharmaceutical composition includes the nonapeptide PQ9, and the amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

6. The pharmaceutical composition according to claim 5, wherein It also includes a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutically acceptable carrier includes one or more of: fillers, wetting agents, disintegrants, binders, and lubricants.

8. The pharmaceutical composition according to claim 6, characterized in that, The preparation form of the pharmaceutical composition is an oral preparation.

9. A health food for relieving constipation, characterized in that, It includes the nonapeptide PQ9 as the active ingredient and food-grade acceptable excipients, and the amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

10. The health food according to claim 9, characterized in that, The dosage form of the health food is beverage, oral liquid, capsule, tablet or granule.

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