Nonapeptide with intestinal peristalsis promoting effect, preparation method and application thereof

Abstract

The present invention discloses a nonapeptide having the function of promoting intestinal peristalsis, a preparation method and application thereof, and belongs to the field of biomedicine technology. The amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln. The peptide segment can be obtained by artificial synthesis or directed enzymatic hydrolysis of wheat peptides. Functional verification shows that the polypeptide has the function of promoting intestinal peristalsis, which is mainly reflected in shortening the time interval between intestinal peristalsis peaks and increasing the intestinal peristalsis promotion rate. The present invention provides a new active substance that promotes intestinal peristalsis, which can be used to prepare medicines for improving intestinal peristalsis disorders or health foods that promote laxative effects, and has good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a nonapeptide capable of promoting intestinal peristalsis, a preparation method thereof and an application thereof. Background Art

[0002] Intestinal peristalsis refers to the peristalsis of worms, especially the wave-like contraction of the intestines. Small intestinal peristalsis is the peristalsis of the small intestine when food is initially digested by the stomach and enters the small intestine, and the small intestine absorbs nutrients; large intestinal peristalsis is the peristalsis of food after digestion and enters the large intestine to wait to be excreted from the body. Intestinal peristalsis dysfunction means that the peristalsis of the intestine is not strong enough or is too slow, resulting in dysfunction of the digestive system. Slowed intestinal peristalsis cannot ensure that toxins in the body are excreted as quickly as possible, resulting in the continuous accumulation of harmful substances in the body. Long-term slowed peristalsis can lead to the occurrence of gastrointestinal diseases such as intestinal obstruction and constipation, and can also cause cardiovascular and cerebrovascular diseases.

[0003] Due to the influence of multiple factors, the exact pathogenesis of peristaltic dysfunction remains unclear. Currently, laxatives are often used to alleviate this symptom. However, laxatives have varying degrees of side effects on the human body. For example, long-term use of anthraquinone laxatives can lead to melanosis coli and increase the risk of colorectal cancer. Therefore, the search for safe and effective natural substances to promote intestinal peristalsis is particularly important.

[0004] Food-derived bioactive peptides can maintain intestinal barrier function by regulating the proliferation and composition of intestinal epithelial cells, alleviating oxidative stress in 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 cellular distribution of tight junction proteins, regulating the diversity and metabolism of commensal microorganisms, comprehensively enhancing intestinal mucosal function, and influencing the inflammatory state of intestinal cells and the activity of immune cells in the underlying lamina propria. Therefore, food-derived bioactive peptides can positively influence gastrointestinal homeostasis by regulating barrier function, immune response, and intestinal microbiota.

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

[0006] The above research subjects are all mixtures of bioactive peptides. Currently, there is insufficient research in this field on small-molecule bioactive peptides that promote intestinal motility. Therefore, it is necessary to analyze the peptide spectrum of proteolytic products and screen out bioactive small-molecule peptides related to the function of promoting intestinal motility. Summary of the Invention

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

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] This study used LC-MS / MS peptide mapping to analyze the peptide sequences in wheat peptides. Molecular docking was then used to investigate the interaction between the peptides and acetylcholine receptors. A candidate peptide was identified by mass spectrometry as Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln (PGYQVHWPQ) with a molecular weight of 1110.52 Da. This peptide was named PQ9. Furthermore, the nonapeptide PQ9 was synthesized and functionally validated, demonstrating a significant pro-intestinal motility effect in an animal model of intestinal motility disorders.

[0010] Therefore, the present invention provides a biologically active nonapeptide PQ9, wherein the amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

[0011] The present invention also provides a method for preparing the nonapeptide PQ9. The nonapeptide PQ9 can be prepared by solid-phase synthesis. The specific method includes: adopting an Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as a solid-phase carrier, sequentially introducing glutamine, proline, tryptophan, histidine, valine, glutamine, tyrosine, glycine, and proline residues to extend the peptide chain from the C-terminus to the N-terminus, and solid-phase synthesizing the nonapeptide PQ9.

[0012] The nonapeptide PQ9 can also be obtained by enzymatic hydrolysis of wheat gluten. Specifically, wheat protein (gluten powder) and water are mixed in a mass ratio of 1:15-20, the pH of the liquid is adjusted to 8.0±0.2, 0.5% alkaline protease of the total weight of gluten powder is added for enzymatic hydrolysis for 30 minutes, and sheared at a speed of 10000-15000 r / min for 15-30 minutes; after the shearing is completed, the pH of the liquid is adjusted to 8.0±0.2, and 1.5% alkaline protease of the total weight of gluten powder is added in sequence for continued enzymatic hydrolysis for 30 minutes, 0.5% neutral protease of the total weight of gluten powder is added for continued enzymatic hydrolysis for 45 minutes, and 0.5% flavor protease of the total weight of gluten powder is added for 30 minutes; after the enzymatic hydrolysis is completed, centrifugation is carried out at a speed of 6000 r / min, and the supernatant is filtered through a membrane with a molecular weight cutoff of 5 kDa. The obtained filtrate contains the nonapeptide PQ9.

[0013] The present invention also provides the use of the nonapeptide PQ9 in the preparation of a drug for improving intestinal motility and / or relieving constipation or a health food for promoting laxative effects. Studies conducted in the present invention have shown that the nonapeptide PQ9 has the effect of promoting intestinal motility. Treatment with the nonapeptide PQ9 in an animal model of intestinal motility disorders can significantly promote intestinal motility and improve intestinal motility disorders. The nonapeptide PQ9 can be used to develop related products for improving intestinal motility disorders.

[0014] Furthermore, the promoting of intestinal motility includes at least one of shortening the intestinal motility peak time interval and increasing the intestinal motility promotion rate.

[0015] The present invention provides a pharmaceutical composition for treating diseases associated with intestinal motility disorders. The active ingredient of the pharmaceutical composition includes the nonapeptide PQ9, wherein the amino acid sequence of the nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln. The diseases associated with intestinal motility disorders may include, but are not limited to, intestinal obstruction and constipation.

[0016] The nonapeptide PQ9 in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that promotes intestinal peristalsis, or it can be used in combination with other active ingredients that have the function of promoting intestinal peristalsis. The active components that promote intestinal peristalsis function can be but are not limited to dietary fiber, probiotics or other protein peptides.

[0017] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier. The present invention uses the nonapeptide PQ9 as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares the preparation according to the preparation preparation method recorded in pharmaceutics.

[0018] Furthermore, the pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder and a lubricant.

[0019] Furthermore, the pharmaceutical composition may be in the form of, but not limited to, an oral preparation. Specifically, the pharmaceutical composition may be in the form of, but not limited to, an oral solution, capsule, microcapsule powder, tablet, granule, or emulsion.

[0020] The present invention also provides a health food for promoting intestinal motility, comprising nonapeptide PQ9 as an active ingredient and food-acceptable excipients. The amino acid sequence of nonapeptide PQ9 is Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln. The health food functions to promote digestion or laxative effects.

[0021] Furthermore, the dosage form of the health food is a beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides a nonapeptide PGYQVHWPQ that has the function of promoting intestinal peristalsis. The peptide segment can be obtained by artificial synthesis or directed enzymatic hydrolysis of wheat peptides. Functional verification of animal models shows that the nonapeptide has the function of promoting intestinal peristalsis, which is mainly reflected in shortening the time interval between intestinal peristalsis peaks and increasing the intestinal peristalsis promotion rate. Moreover, the nonapeptide PGYQVHWPQ is a natural peptide segment with high biosafety. Therefore, it can be used to prepare drugs for improving intestinal peristalsis disorders or health foods that promote laxative effects. The present invention provides a new solution for improving intestinal peristalsis disorders and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the secondary mass spectrum of the nonapeptide PQ9. 1:1 represents the numbering; P / G / Y / Q / V / H / W / P / Q represents the nonapeptide sequence; the ordinate Intensity (%) represents the relative abundance; b2 represents the second fragment ion produced by the N-terminal cleavage of the peptide; y2 represents the second fragment ion produced by the C-terminal cleavage of the peptide; y3 represents the third fragment ion produced by the C-terminal cleavage of the peptide; b4 represents the fourth fragment ion produced by the N-terminal cleavage of the peptide; b5 represents the fifth fragment ion produced by the N-terminal cleavage of the peptide; y4 represents the fourth fragment ion produced by the C-terminal cleavage of the peptide; y5 represents the fifth fragment ion produced by the C-terminal cleavage of the peptide; b6 represents the sixth fragment ion produced by the N-terminal cleavage of the peptide; y6 represents the sixth fragment ion produced by the C-terminal cleavage of the peptide; b7 represents the seventh fragment ion produced by the N-terminal cleavage of the peptide; y7 represents the seventh fragment ion produced by the C-terminal cleavage of the peptide; b8 represents the eighth fragment ion produced by the N-terminal cleavage of the peptide; y8 represents the eighth fragment ion produced by the C-terminal cleavage of the peptide.

[0025] Figure 2Schematic diagram of the binding interaction between nonapeptide PQ9 and CHRM1.

[0026] Figure 3 Comparison of intestinal motility images recorded by stereomicroscope after the nonapeptide PQ9 and LH9 acted on the zebrafish model.

[0027] Figure 4 To compare the effects of nonapeptide PQ9 and LH9 on the intestinal peristalsis time interval of zebrafish, * indicates significant difference compared with the blank group (NC), * indicates P <0.05; # indicates significant difference compared with constipation model group (MC); ## indicates P <0.01.

[0028] Figure 5 Comparison of the effects of nonapeptide PQ9 and LH9 on the zebrafish model using in vivo fluorescence microscopy.

[0029] Figure 6 To compare the effects of nonapeptide PQ9 and LH9 on the intestinal motility promotion rate of zebrafish, * indicates significant difference compared with the blank group (NC), * indicates P <0.05, ** indicates P <0.01.

[0030] Figure 7 Comparison of intestinal motility images recorded by stereomicroscope after the nonapeptide PQ9 and wheat peptide acted on the zebrafish model.

[0031] Figure 8 To compare the effects of nonapeptide PQ9 and wheat peptide on the time interval of intestinal peristalsis in zebrafish, * indicates significant difference compared with the blank group (NC), * indicates P <0.05; # indicates significant difference compared with constipation model group (MC); ## indicates P <0.01, ### indicates P <0.001.

[0032] Figure 9 Comparison of the effects of nonapeptide PQ9 and wheat peptide on the zebrafish model using in vivo fluorescence microscopy.

[0033] Figure 10 To compare the effects of nonapeptide PQ9 and wheat peptide on the intestinal motility promotion rate of zebrafish, * indicates significant difference compared with the blank group (NC), *** indicates P <0.001. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0036] Wheat protein (gluten) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; and flavor protease was purchased from Angel Enzyme (Yichang) Co., Ltd.

[0037] Example 1: Screening of active peptides

[0038] 1. Preparation of wheat peptide (glutamine peptide)

[0039] Wheat protein (gluten) and water were added into the enzymatic hydrolysis reaction tank at a ratio of 1:15, the pH of the liquid was adjusted to 8.0±0.2, and alkaline protease (derived from Bacillus licheniformis) at a concentration of 0.5% of the total weight of gluten was added for enzymatic hydrolysis for 30 minutes. Then, the shearing machine was turned on with a shearing speed of 15,000 r / min for 30 minutes.

[0040] After the shearing is completed, the pH of the feed solution is adjusted to 8.0±0.2, and alkaline protease (derived from Bacillus licheniformis) at a concentration of 1.5% of the total weight of gluten is added to continue enzymatic hydrolysis for 30 min. After the end, neutral protease (derived from Bacillus subtilis) at a concentration of 0.5% of the total weight of gluten is added to continue enzymatic hydrolysis for 45 min. After the end, flavor protease at a concentration of 0.5% of the total weight of gluten is added to continue enzymatic hydrolysis for 30 min.

[0041] After the enzymatic hydrolysis, the mixture was separated by a disc centrifuge (speed 6000 r / min), and the supernatant was filtered through a membrane with a molecular weight cutoff of 5 kDa. After filtration, the supernatant was sterilized and spray-dried to obtain wheat peptide (glutamine peptide) powder.

[0042] 2. Screening of active peptides

[0043] LC-MS / MS peptide spectrum analysis was performed on wheat peptides, and the peptide segments obtained were sorted according to the screening conditions. Then molecular docking was further used to determine the final theoretical effective peptide segments based on the scores. The specific analysis process is as follows:

[0044] (1) Wheat peptide sequence identification

[0045] Wheat peptide samples were dissolved in NH₄HCO₃ solution, dithiothreitol solution was added, and the mixture was reduced in a 56°C water bath for 1 hour. Iodoacetamide solution was then added and allowed to react for 40 minutes in the dark. After desalting, the solvent was evaporated to dryness, and the sample was dissolved in 10 μL of mobile phase A (0.1% formic acid) and transferred to a liquid chromatography injection vial. LC-MS / MS analysis was then performed.

[0046] 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 to 8% B; 2–45 min, 8% B to 40% B; 45–55 min, 40% B to 60% B; 55–56 min, 60% B to 95% B; 56–66 min, 95% B.

[0047] Mass spectrometry conditions: Full scan MS was performed using an Orbitrap primary scan with a scan range of 100–1500 m / z, a resolution of 70,000, a maximum ion introduction time of 100 ms, and an automatic gain control of 3×106. High-energy collisional dissociation was used to fragment the top 20 precursor ions that met the tandem (MS / MS) fragmentation conditions and scanned with an Orbitrap scan with a resolution of 17,500, a maximum ion introduction time of 50 ms, and an automatic gain control of 1×106. 5 The raw data obtained by mass spectrometry were used to analyze the peptide sequence using PEAKSStudio De novo (de novo sequencing) software.

[0048] (2) Screening of active peptides with potential to promote intestinal motility

[0049] The enteric nervous system is the largest component of the peripheral nervous system and can autonomously control intestinal function independently of the central nervous system. Patients with severe constipation, especially those who take stimulant laxatives for a long time, often show enteric nervous system dysfunction, including a decrease in the number of colonic neurons and damaged cell populations. In intestinal neurons, activation of muscarinic acetylcholine receptors (mAChRs) can lead to calcium influx, triggering a series of intracellular signaling, thereby participating in the regulation of intestinal motility, secretion, and other physiological processes. CHRM1 is one of the mAChR subtypes. Previous studies have found that wheat peptides (mixtures) can effectively promote intestinal motility and activate CHRM1. Therefore, molecular docking of wheat peptide fragments with CHRM1 was performed to screen small molecule peptide fragments that may promote intestinal motility.

[0050] Specifically, the average local confidence (ALC) is greater than 95%, the peak area is greater than 2×10 6, PeptideRanker scores greater than 0.8 were screened to obtain qualified peptides. The peptides were then docked with the M1-muscarinic acetylcholine receptor (CHRM1) for molecular docking.

[0051] First, the crystal structure of CHRM1 (5CXV) was downloaded from the PDB protein database. Using Discovery Studio software, water molecules were removed from the receptor target and hydrogen atoms were added to define its active site. The structures of the selected wheat peptide segments were constructed using Discovery Studio, and their energies were minimized using the CHARMm force field to define these peptides as ligands. CDOCKER docking of the constructed peptides with CHRM1 was performed to simulate the binding mode, site, and interacting amino acid residues with the lowest binding energy and highest binding strength. Screening was performed based on binding energy and number of hydrogen bonds. Finally, two nonapeptides (PGYQVHWPQ and LNPFPFEEH) were identified, as shown in Table 1.

[0052] Table 1. Peptides from wheat peptides with potential intestinal motility-promoting activity

[0053] 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

[0054] The secondary mass spectrum of the nonapeptide PQ9 is as follows Figure 1 As shown in the figure, the fragment ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z type ions break to form d, v, and w type ions respectively. In addition, there are internal ions formed by the breakage of the two ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. 556.268 m / z is the [M+H] + The ion signal, charge number (z) was 2, and the molecular weight was 1110.52 Da, which was basically consistent with the nonapeptide PGYQVHWPQ. The nonapeptide was further analyzed by secondary mass spectrometry using the in-source collision-induced dissociation technique (e.g. Figure 1 ), the primary structure of the nonapeptide was determined to be Pro-Gly-Tyr-Gln-Val-His-Trp-Pro-Gln.

[0055] The 2D and 3D images of molecular docking of PQ9 and CHRM1 are shown in Figure 2. Figure 2Chemical bond analysis revealed that PQ9 is primarily bound to CHRM1 through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and hydrophobic interactions (π-alkyl groups), 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.

[0056] The molecular docking results show that the nonapeptide PQ9 can bind to CHRM1 at the molecular docking level, thereby activating CHRM1 to regulate intestinal motility.

[0057] Shenzhen Borunsida Biotechnology Co., Ltd. was commissioned to synthesize the peptides PGYQVHWPQ and LNPFPFEEH with a purity of ≥98% for subsequent functional verification.

[0058] Example 2: Comparison of the effects of nonapeptides PQ9 and LH9 on intestinal peristalsis in a zebrafish intestinal peristalsis disorder model

[0059] 1. Comparison of the effects of nonapeptide PQ9 and LH9 on the intestinal peristalsis interval of zebrafish

[0060] This example uses wild-type AB strain zebrafish for research. The time interval between two intestinal peristalsis peaks of zebrafish is measured to evaluate the intestinal peristalsis promoting effect of the sample.

[0061] AB zebrafish (4 months old) were housed in recirculating system water at 28 ± 0.5°C and a pH of 7.0-7.5, with a 14-h light / 10-h dark photoperiod and brine shrimp fed twice daily. Healthy adult zebrafish were placed in a breeding tank at a 1:1 ratio of male to female on the evening of the previous day, with the water level approximately 2 / 3 full and separated by a partition. The next morning, around 8:00 AM, the partition was removed, allowing the fish to fertilize freely and harvest embryos at 0 hpf. After cleaning the embryos from impurities, they were placed in a Petri dish containing methylene blue system water and incubated in a constant-temperature incubator at 28 ± 0.5°C. After 24 hours, phenylthiourea (PTU) was added to inhibit melanin synthesis.

[0062] Zebrafish at 5 dpf were plated in six-well plates and divided into four groups, with 20 per well. The groups were blank (NC), model (MC), PQ9 (5 μg / mL), and LH9 (5 μg / mL). The blank group received an equal volume of system water, the model group received 10 μg / mL ropiramide hydrochloride, and the sample group received the corresponding dose of sample and ropiramide hydrochloride. After 24 hours of incubation at 28 ± 0.5°C, intestinal peristalsis was recorded using a stereomicroscope. After the experiment, the time interval between two intestinal peristalsis peaks was recorded by repeatedly viewing the videos.

[0063] This study used Graphpad Prism 8.0 software for statistical analysis and plotting. All data were expressed as mean ± standard error. One-way analysis of variance was used to compare differences between groups (compared with the NC group, * P <0.05,** P <0.01,*** P <0.001; compared with the MC group, # P <0.05,## P <0.01,### P <0.001).

[0064] The results are as follows Figure 3 and Figure 4 As shown, the bowel movement interval in the MC group was significantly increased compared with the NC group ( P <0.05), indicating that the intestinal motility disorder model was successfully established. Compared with the MC group, LH9 treatment shortened the intestinal motility interval, but it did not reach a significant level ( P >0.05), while PQ9 treatment can significantly shorten the intestinal peristalsis interval ( P <0.01), indicating that PQ9 has a significant effect on the time interval between bowel movements.

[0065] 2. Comparison of the effects of nonapeptide PQ9 and LH9 on the intestinal motility promotion rate of zebrafish

[0066] The fluorescent dye Nile red is not interfered with by other tissues in the zebrafish body and is not absorbed. By quantifying Nile red in the zebrafish gastrointestinal tract, the intestinal motility promoting effect can be evaluated.

[0067] 5 dpf zebrafish were selected and stained with Nile red dye. The zebrafish were cultured in the dark for 16 hours. The dye was then washed away with system water, and the zebrafish were transferred to a 6-well plate and divided into 3 groups, with 20 fish in each well. The specific groups were blank group, PQ9 group (5 μg / mL) and LH9 group (5 μg / mL). The blank group was added with the same volume of system water, and the sample group was added with samples of the corresponding dose. After culturing in the dark for 24 hours, the same magnification, fluorescence intensity, exposure time and fluorescence gain were used, and pictures were taken under a stereo fluorescence microscope. The total fluorescence signal of the zebrafish intestine was analyzed using NIS-Elements D 3.10 advanced image processing software. The effect of the sample on the intestinal motility of the zebrafish was judged by the amplitude of the fluorescence intensity reduction before and after. The calculation method is:

[0068] Intestinal motility promotion rate = (S blank control group - S sample group) / S blank control group × 100%

[0069] The results are as follows Figure 5 and Figure 6 As shown in Figure 2, compared with the NC group, both nonapeptide PQ9 and LH9 could significantly increase the rate of intestinal motility promotion ( P <0.01 and P <0.05), indicating that both PQ9 and LH9 had significant effects on promoting intestinal motility, but the effect of LH9 was lower than that of PQ9.

[0070] The comprehensive results of intestinal peristalsis peak time interval and intestinal peristalsis promotion rate show that nonapeptide PQ9 has the effect of promoting intestinal peristalsis.

[0071] Example 3: Comparison of the effects of nonapeptide PQ9 and wheat peptides on intestinal peristalsis in a zebrafish intestinal peristalsis disorder model

[0072] 1. Comparison of the effects of nonapeptide PQ9 and wheat peptide on the time interval of intestinal peristalsis in zebrafish

[0073] The experimental method was the same as that used in the study “Comparison of the effects of nonapeptides PQ9 and LH9 on the time interval between intestinal motility in zebrafish”. The animals were divided into 8 groups, with 20 per well, including a blank group (NC), a model group (MC), a low-dose (10 μg / mL, WP-10) and high-dose (100 μg / mL, WP-100) wheat peptide groups, and the lowest-dose (0.5 μg / mL, PQ9-0.5), low-dose (1 μg / mL, PQ9-1), medium-dose (5 μg / mL, PQ9-5), and high-dose (10 μg / mL, PQ9-10) PQ9 groups.

[0074] The results are as follows Figure 7 and Figure 8 As shown, the bowel movement interval in the MC group was significantly increased compared with the NC group ( P<0.05), indicating that the intestinal peristalsis disorder model was successfully established. Compared with the MC group, 10 μg / mL wheat peptide shortened the intestinal peristalsis interval, but did not reach a significant ( P >0.05), when the concentration of wheat peptide was 100 μg / mL, it could significantly shorten the intestinal peristalsis time interval ( P <0.01). When the concentration of nonapeptide PQ9 was 5 μg / mL and 10 μg / mL, it could significantly shorten the intestinal peristalsis interval ( P <0.001). Based on the above data, it can be seen that the effect of promoting intestinal peristalsis by nonapeptide PQ9 is about 20 times that of wheat peptide from the perspective of intestinal peristalsis time interval.

[0075] 2. Comparison of the effects of nonapeptide PQ9 and wheat peptide on the intestinal motility promotion rate of zebrafish

[0076] The experimental method was the same as that used in the study “Comparison of the effects of nonapeptides PQ9 and LH9 on the promotion of intestinal motility in zebrafish.” The animals were divided into seven groups, with 20 per well, including a blank group, low-dose (10 μg / mL) and high-dose (100 μg / mL) wheat peptide groups, and low-dose (0.5 μg / mL), low-dose (1 μg / mL), medium-dose (5 μg / mL), and high-dose (10 μg / mL) PQ9 groups.

[0077] The results are as follows Figure 9 and Figure 10 As shown in Figure 2, compared with the NC group, 10 μg / mL wheat peptide increased the intestinal motility promotion rate, but did not reach a significant level ( P >0.05), when the concentration of wheat peptide was 100 μg / mL, it could significantly increase the intestinal motility promotion rate ( P <0.001). When the concentration of nonapeptide PQ9 was 1-10 μg / mL, it could significantly increase the rate of promoting intestinal motility ( P <0.001). Based on the above data, it can be seen that the effect of promoting intestinal peristalsis by nonapeptide PQ9 is about 100 times that of wheat peptide in promoting intestinal peristalsis.

[0078] In summary, the present invention screened out the nonapeptide PGYQVHWPQ from wheat peptides through mass spectrometry identification and molecular docking of peptide segments. The peptide segment showed efficient intestinal motility-promoting activity in the intestinal motility disorder model, which was mainly reflected in shortening the intestinal motility peak time interval and improving the intestinal motility promotion rate. The present invention provides a theoretical basis for the development of new active substances that promote intestinal motility, and can be applied to drugs that improve intestinal motility disorders or health foods that promote laxative effects. The nonapeptide PGYQVHWPQ can be used alone to prepare drugs that improve intestinal motility disorders or health foods that promote laxative effects, and can also be compounded with other active ingredients that have the function of promoting intestinal motility. The active components that promote intestinal motility function 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 fields.

Claims

1. A biologically active 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 method for preparing the nonapeptide PQ9 according to claim 1, wherein: The nonapeptide PQ9 is prepared by solid phase synthesis.

3. Use of the nonapeptide PQ9 according to claim 1 in the preparation of a drug for improving intestinal motility disorders and / or relieving constipation or a health food for promoting laxative effects, wherein: The nonapeptide PQ9 has the effect of promoting intestinal peristalsis.

4. The use according to claim 3, characterized in that The promoting of intestinal peristalsis includes at least one of shortening the intestinal peristalsis peak time interval 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 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 Also included are pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, wherein The pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder and a lubricant.

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

9. A health food for promoting laxative effect, characterized in that: The invention comprises nonapeptide PQ9 as an active ingredient and excipients acceptable in food science, wherein 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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