Fructus cannabis extracted polypeptide with hypoglycemic function

Abstract

The invention relates to a polypeptide with a blood sugar reducing effect. The amino acid sequence of the polypeptide is selected from one of IPVIA, IPVA, IPTA, IPVVA, IPVPA, VVPL, VPKPL, VPSL, IPPA, VIPL, VPAL, VPVVL, VPQ, VPIIL, HAYVFGVEDTLNALESGAIETL WENLDINR or YVFGVEDTLNALESGAIETL IVWENLDINR. The invention further relates to a preparation method of the polypeptide.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a polypeptide extracted from hemp seed and its application in reducing blood sugar. Background Art

[0002] Diabetes is widely spread around the world. It is mainly caused by impaired insulin metabolism or insufficient secretion and is characterized by high blood glucose concentration. With the continuous study of the pathogenesis of diabetes, DPP-IV has gradually become a new target for the treatment of diabetes. DPP-IV is a 766-amino acid serine protease that is ubiquitous in the human body, especially expressed in many tissues such as the lungs, brain and kidneys. Based on in vitro studies, it was found that this enzyme preferentially hydrolyzes Xaa-Pro (proline) or Xaa-Ala (alanine) at the N-terminus of the polypeptide, where Xaa represents any of the 20 natural amino acids. The proline residue is present in the second position of its N-terminus. As an evolutionarily conservative strategy, it can protect several bioactive peptides from general proteolytic attacks. The second position of the N-terminus of GLP-1 is Ala, which will be cleaved by DPP-IV after release and quickly enzymatically inactivated, thereby losing its function of promoting blood sugar lowering. DPP-IV inhibitors inactivate DPP-IV, increase the level of GLP-1 in the body, and produce the effect of regulating blood sugar. Therefore, the research and development of DPP-IV inhibitors has become an important direction for the prevention and treatment of type 2 diabetes and obesity.

[0003] In recent years, bioactive peptides from natural sources have become a research hotspot in the development of drugs or functional foods due to their mild effects, clear functions, high safety, and few side effects. Bioactive peptides with DPP-IV inhibitory effects, as a natural DPP-IV inhibitor, have the advantages of mild effects, clear functions, high safety, and few side effects. They have become a research hotspot in recent years and have broad application prospects as drugs or functional foods. Summary of the invention

[0004] Hemp seed is the dried mature seed of the plant Cannabis sativa L. of the Moraceae family. It has the properties of medicine and food. It is cultivated in various parts of my country. There are also semi-wild ones. It is widely distributed in Northeast my country, North China, East China, Central and South China. Hemp seed is flat in nature, sweet in taste, has the effect of moistening the intestines and facilitating bowel movements, and is rich in protein, vitamins, lecithin, volatile oil, and trace elements such as calcium and magnesium.

[0005] The purpose of the present application is to provide an active polypeptide derived from hemp seed with hypoglycemic function.

[0006] Specifically, the present application provides a polypeptide, whose amino acid sequence is selected from any one of IPVIA (Seq ID No.1), IPVA (Seq ID No.2), IPTA (Seq ID No.3), IPVVA (Seq ID No.4), IPVPA (Seq ID No.5), VVPL (Seq ID No.6), VPKPL (Seq ID No.7), VPSL (Seq ID No.8), IPPA (Seq ID No.9), VIPL (Seq ID No.10), VPAL (Seq ID No.11), VPVVL (Seq ID No.12), VPIIL (Seq ID No.13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.15) or VPQ.

[0007] The present application provides a nucleic acid sequence encoding the polypeptide.

[0008] The present application provides a composition, which includes one or more polypeptides having an amino acid sequence of IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15) or VPQ.

[0009] Preferably, the composition provided in the present application further comprises a pharmaceutically, food or health product acceptable carrier, excipient or second active ingredient.

[0010] Preferably, the second active ingredient in the composition provided by the present application is selected from one or more of a DPP-IV inhibitor, a SGLT-2 inhibitor, a GLP-1 receptor agonist, an insulin secretagogue, an α-glucosidase inhibitor, an angiotensin converting enzyme inhibitor, an angiotensin receptor antagonist, a calcium channel blocker or a β-receptor blocker.

[0011] The present application provides amino acid sequences selected from IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15). Use of any one of the polypeptides No. 15) or VPQ or a composition comprising one or more of the polypeptides in the preparation of drugs, special medical purpose foods and health foods for preventing, treating or improving hyperglycemia and human-related diseases caused by hyperglycemia.

[0012] The present application provides a method for preventing, treating or improving hyperglycemia and human-related diseases caused by hyperglycemia, the method comprising administering to a subject an effective amount of a polypeptide, wherein the amino acid sequence of the polypeptide is selected from any one of IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15) or VPQ.

[0013] Furthermore, the polypeptide provided in the present application is extracted from hemp seed.

[0014] Effects of the Invention

[0015] The polypeptide provided in the present application has an inhibitory effect on DPP-IV, can significantly promote the secretion of GLP-1 and PYY, improve glucose tolerance and improve insulin resistance symptoms, and achieve the effect of lowering blood sugar and promoting blood sugar metabolism balance. At the same time, compared with the common chemical drugs that inhibit DPP-IV in the prior art, the polypeptide and its composition provided in the present application are polypeptide sequences present in the medicinal and edible crops hemp seeds, which have almost no side effects when eaten and are safer for human bodies. Compared with other hypoglycemic peptides in the prior art, such as GLP-1 receptor agonists exenatide, lixisenatide, liraglutide, albiglutide, and dulaglutide, because of the large number of amino acids and long polypeptide sequences, if you want to ensure the efficacy, you need to choose the injection method, and the polypeptide in the present invention is a 2-8 peptide that can avoid gastrointestinal digestion, so it can be taken orally to achieve a hypoglycemic effect, and it is safer and more convenient to take. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a schematic diagram of the basic process of establishing a hemp seed peptide database with hypoglycemic function.

[0017] Figure 2 Shown is a schematic diagram of the results of the inhibition type determination of the polypeptide in Example 3.

[0018] Figure 3Shown is a schematic diagram of the results of the DPP-IV inhibitory activity assay in Example 4.

[0019] Figure 4 Shown is a schematic diagram of the GLP-1 concentration measurement results in Example 4.

[0020] Figure 5 Shown is a schematic diagram of the PYY concentration measurement results in Example 4. DETAILED DESCRIPTION

[0021] The present application is further described in detail below in conjunction with specific implementation methods. The examples given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0022] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0023] In this application, "peptide" is a compound formed by α-amino acids linked together by peptide bonds, and is an intermediate product of protein hydrolysis. A compound formed by the dehydration condensation of two amino acid molecules is called a dipeptide, and by analogy, there are tripeptides, tetrapeptides, pentapeptides, etc. "Polypeptide" refers to a peptide composed of three or more amino acid molecules.

[0024] In this application, "DPP-IV" refers to DPP-4, also known as dipeptidyl peptidase IV, which is a biologically active enzyme in the human body that can break down a protein called GLP-1 in the body, rendering GLP-1 inactive. GLP-1 is a hormone secreted by the intestine that stimulates insulin secretion, inhibits glucagon secretion, and inhibits gastric emptying. The DPP-4 inhibitor class of hypoglycemic drugs used in clinical practice inhibits the DPP-4 enzyme, thereby increasing the level of GLP-1 in the body to achieve a hypoglycemic effect. This class of drugs includes sitagliptin, linagliptin, alogliptin, saxagliptin, vildagliptin, etc.

[0025] In this application, "GLP-1" refers to glucagon-like peptide-1, which is an important incretin hormone secreted by intestinal L cells. Its main physiological effects in the body include stimulating the secretion and release of insulin, inhibiting the secretion of glucagon, promoting the proliferation of pancreatic β cells and inhibiting their apoptosis, inhibiting gastric emptying, and promoting the production of satiety. GLP-1 has a good therapeutic prospect for diabetes and obesity. Since GLP-1 is quickly degraded by dipeptidyl peptidase IV in the body and has a short plasma half-life, its clinical application is limited.

[0026] In this application, the "chromogenic substrate method" is to use the absorbance change of the chromogenic substance to estimate the content of the substance being measured. The basic principle is: first, a compound containing a certain active enzyme cleavage point is artificially synthesized, and a chromogenic substance (such as p-nitroaniline, PNA) is connected to this compound. The plasma to be tested contains an active enzyme or an active enzyme is added to the sample. The chromogenic substance in the compound can be cleaved, causing the sample to be tested to change color. Based on this color change, the content of the substance being tested can be estimated.

[0027] The present application provides a polypeptide extracted from hemp seed. Specifically, the polypeptide provided in the present application inhibits the enzymatic activity of DPP-IV, so that GLP-1 will not be rapidly degraded by DPP-IV, thereby prolonging the action time of GLP-1 and enhancing its positive effect in the process of lowering blood sugar. This polypeptide has inhibitory activity against DPP-IV.

[0028] In a specific embodiment, the polypeptide is selected from any one of IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15) or VPQ (Seq ID No. 16).

[0029] In the aforementioned polypeptide sequences, capital letters such as I, P, V, A, K, D, S, T, and W each represent an amino acid or its amino acid residue. The correspondence between the capital letters and the amino acids is shown in Table 1.

[0030] Table 1 Amino acids and their corresponding capital letters

[0031]

[0032]

[0033] The present application also provides a nucleic acid sequence encoding the polypeptide.

[0034] In a specific embodiment, the polypeptide can be produced by chemical synthesis. In some embodiments, the polypeptide can be produced by biosynthesis. In some embodiments, the polypeptide can be extracted from food or obtained by enzymatic hydrolysis.

[0035] The present application also provides a composition, which comprises one or more polypeptides having an amino acid sequence of IPVIA (Seq ID No.1), IPVA (Seq ID No.2), IPTA (Seq ID No.3), IPVVA (Seq ID No.4), IPVPA (Seq ID No.5), VVPL (Seq ID No.6), VPKPL (Seq ID No.7), VPSL (Seq ID No.8), IPPA (Seq ID No.9), VIPL (Seq ID No.10), VPAL (Seq ID No.11), VPVVL (Seq ID No.12), VPIIL (Seq ID No.13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.15) or VPQ.

[0036] In some specific embodiments, the composition is a pharmaceutical composition, a functional food or a health product.

[0037] In a specific embodiment, the composition provided herein comprises one, two or more polypeptides provided herein.

[0038] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0039] In a specific embodiment, preferably, the composition provided in the present application further comprises a pharmaceutically, food or health product acceptable carrier, excipient or second active ingredient.

[0040] Hyperglycemia is a metabolic disorder caused by a significant increase in blood sugar. Human-related diseases caused by hyperglycemia include chronic diseases caused by long-term hyperglycemia, such as diabetes, retinopathy, kidney disease, diabetic nephropathy, diabetic retinopathy, atherosclerosis, peripheral neuropathy, autonomic neuropathy, etc.; they also include acute and severe metabolic disorders caused by a significant increase in acute blood sugar, such as diabetic ketoacidosis, hyperosmolar hyperglycemic syndrome, etc.

[0041] In a specific embodiment, the polypeptide provided in the present application or a composition comprising the polypeptide can inhibit DPP-IV activity, increase the level of GLP-1 in the body, produce the effect of regulating blood sugar, and can be applied to the development of drugs, special medical purpose foods and health foods for hyperglycemia and human-related diseases caused by hyperglycemia.

[0042] In a specific embodiment, as an inhibitor of DPP-IV, the polypeptide or a composition comprising the polypeptide can be administered to a human body for the prevention, treatment and improvement of hyperglycemia and its related diseases, especially type 2 diabetes. In a specific embodiment, the polypeptide or a composition comprising the polypeptide can reduce the risk of cardiovascular diseases caused by factors such as type 2 diabetes and hyperglycemia.

[0043] In a specific embodiment, the polypeptide or a composition comprising the polypeptide can be administered to a human body for the prevention, treatment and improvement of type 1 diabetes.

[0044] In a specific embodiment, the polypeptide or a composition comprising the polypeptide can be administered to the human body for the prevention, treatment and improvement of physiological hyperglycemia (e.g., improper diet, stress, drug factors, poor mental state, etc.), pathological hyperglycemia (infection, reactive hyperglycemia, myocardial infarction, cerebral infarction and other diseases, etc.) or other conditions of hyperglycemia.

[0045] In a specific embodiment, the second active ingredient in the composition provided by the present application is a polysaccharide, a polyphenol, a flavonoid, or a terpene compound.

[0046] Preferably, the second active ingredient in the composition provided by the present application is selected from one or more of a DPP-IV inhibitor, a SGLT-2 inhibitor, a GLP-1 receptor agonist, an insulin secretagogue, an α-glucosidase inhibitor, an angiotensin converting enzyme inhibitor, an angiotensin receptor antagonist, a calcium channel blocker or a β-receptor blocker.

[0047] Specifically, the composition can be prepared in the following form: the polypeptide is mixed with a pharmaceutically acceptable carrier, for example, to obtain an oral preparation, such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), etc., parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drops), external preparations (e.g., skin preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalants), eye drops, etc. In addition, these preparations can be used as controlled release preparations (e.g., sustained release microcapsules), such as immediate release preparations, sustained release preparations, etc. Such preparations can be obtained by conventional preparation methods used in the art.

[0048] Specifically, examples of the above-mentioned pharmaceutically acceptable carriers include excipients (e.g., starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, alginic acid, gelatin, polyvinyl pyrrolidone, etc.), lubricants (e.g., magnesium stearate, calcium stearate, talc, etc.), disintegrants (e.g., carboxymethyl cellulose calcium, talc, etc.), diluents (e.g., water for injection, saline, etc.), additives (e.g., stabilizers, preservatives, colorants, flavorings, dissolution aids, emulsifiers, buffers, isotonic agents, etc.), and the like.

[0049] Specifically, examples of the above-mentioned pharmaceutically acceptable excipients include diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, etc.), absorbents (e.g., calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, etc.), wetting agents (e.g., water, ethanol, etc.), binders (e.g., hydroxypropyl methylcellulose (HPMC), polyvidone (PVP), starch slurry, syrup, etc.), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, cross-linked polyvinylpyrrolidone, etc.), lubricants (e.g., magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, micro powder silica gel), colorants (e.g., titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red), coating materials (e.g., acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate, etc.), and the like.

[0050] In a specific embodiment, the amino acid sequence is IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15). Use of a polypeptide of No. 15) or VPQ or a composition comprising the polypeptide in the preparation of a drug, a special medical purpose food or a functional food for preventing, treating or improving hyperglycemia and human-related diseases caused by hyperglycemia.

[0051] The present application also provides a method for preventing or treating hyperglycemia, the method comprising administering to a subject an effective amount of a polypeptide, wherein the amino acid sequence of the polypeptide is selected from one of IPVIA (Seq ID No.1), IPVA (Seq ID No.2), IPTA (Seq ID No.3), IPVVA (Seq ID No.4), IPVPA (Seq ID No.5), VVPL (Seq ID No.6), VPKPL (Seq ID No.7), VPSL (Seq ID No.8), IPPA (Seq ID No.9), VIPL (Seq ID No.10), VPAL (SeqID No.11), VPVVL (Seq ID No.12), VPIIL (Seq ID No.13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.15) or VPQ.

[0052] The present application also provides a method for improving hyperglycemia, the method comprising administering to a subject an effective amount of a polypeptide, wherein the amino acid sequence of the polypeptide is selected from one of IPVIA (Seq ID No.1), IPVA (Seq ID No.2), IPTA (SeqID No.3), IPVVA (Seq ID No.4), IPVPA (Seq ID No.5), VVPL (Seq ID No.6), VPKPL (Seq ID No.7), VPSL (Seq ID No.8), IPPA (Seq ID No.9), VIPL (Seq ID No.10), VPAL (Seq ID No.11), VPVVL (Seq ID No.12), VPIIL (Seq ID No.13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.15) or VPQ.

[0053] The polypeptides described herein or compositions comprising the polypeptides can be administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys). The administration method can be oral or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, instillation, intracerebral, rectal, vaginal, intraperitoneal, etc.).

[0054] The dosage of the polypeptide of the present application to be administered to a subject varies depending on the administration route, symptoms, patient age, patient weight, etc., and can be determined by a clinician.

[0055] The polypeptides or compositions comprising the polypeptides involved in the present application may also be used together with other existing known drugs for treating hyperglycemia and type 2 diabetes. When used together, there is no restriction on the administration time of each drug, two or more different drugs may be administered simultaneously, and each drug may be administered at different times. The dosage of a known drug may be determined according to the number of administrations used clinically, and may be appropriately selected according to the patient being administered, the route of administration, etc.

[0056] In a specific embodiment, the above polypeptide is extracted from hemp seed.

[0057] Example

[0058] Example 1 Establishment of a database of hemp seed polypeptides with hypoglycemic function

[0059] Hemp seed sample preparation: The hemp seeds were incubated for a certain period of time for germination. The seed incubation conditions were a 15 cm culture dish. After adding 6 layers of filter paper, 20 mL of sterile water was added. The dish was exposed to light and 10 mL of sterile water was added every 24 h. Samples were taken at 0 h, 12 h, 24 h, 48 h and 72 h of incubation.

[0060] Transcriptome: The Trizol method was used to extract mRNA from the above-mentioned samples, and transcriptome sequencing technology was used to perform hemp seed transcriptome sequencing (RNA-seq denovo) to obtain the amino acid sequence information of hemp seed protein.

[0061] Protein group: The hemp seed protein of the samples collected at 12, 24, 48, and 72 hours was extracted using a plant seed protein extraction kit, and the protein content in the protein extract was quantified using Nanodrop. SDS-page gel was run, and in-gel enzymatic digestion was performed with trypsin. Then, the samples were detected using NanoLC-LTQ-Orbitrap Velos liquid spectrometer. The protein types annotated at 12, 24, 48, and 72 hours were 27389, 28483, 32663, and 24049, respectively.

[0062] Hemp seed protein database: Maxquant (https: / / www.maxquant.org / ) proteomics data processing software was used to analyze the hemp seed protein data collected by the proteome, and the protein amino acid sequence information database obtained by the transcriptome was matched and identified. The number of protein types identified at 0h and 72h were 2871 and 2451, respectively, and the hemp seed protein database was established.

[0063] Hemp seed polypeptide database: The protein sequences obtained from the above protein database were imported into the existing public protease cleavage databases (PeptideCutter, PeptideMass, BIOPEP, etc.) for simulated enzyme cleavage. The enzymes used for simulated enzyme cleavage and the number of polypeptides produced by the corresponding simulated enzyme cleavage of 0h protein are shown in Table 2. After deleting the duplicate sequences, the proteins identified at 0h produced a total of 369,127 different polypeptide sequences after simulated enzyme cleavage.

[0064] Table 2 Simulated enzyme cleavage information of hemp seed protein

[0065]

[0066] Database of hemp seed hypoglycemic active peptides:

[0067] The polypeptides in the hemp seed polypeptide database established above were imported into the Biopep database (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep / ) to perform functional annotations on them, so as to screen the hemp seed active polypeptides with hypoglycemic function. The annotation results are shown in Table 3. Among them, the hemp seed protein of germination 0h produced 283 and 14 DPP IV and alpha-glucosidase inhibitory active polypeptide sequences with different amino acid sequences respectively after simulated enzyme digestion, and the number of sequences was 65201 and 5399 respectively.

[0068] Table 3 Peptide activity information of hemp seed peptide database

[0069]

[0070]

[0071] The results shown in Table 3 show that hemp seed is rich in hypoglycemic, antihypertensive, anti-inflammatory and antioxidant active polypeptides.

[0072] Example 2 Screening of polypeptides with DPP IV inhibitory activity based on structural similarity

[0073] The molecular similarity calculation method in the RDKit toolkit was used to calculate the similarity between the hemp seed polypeptide sequence obtained in the above hemp seed polypeptide database and the hypoglycemic peptides (VPL, IPI) and GLP-1 structures (the similarity value is between 0 and 1, and the larger the value, the higher the similarity), and the polypeptides with similar structures to VPL, IPI and GLP-1 (similarity greater than 0.85) were screened out, the number of which was 91, 26 and 146, respectively, and the specific amino acid sequence information is shown in Table 4. The above polypeptide sequences constitute the hemp seed hypoglycemic active polypeptide database.

[0074] Table 4 Hemp seed polypeptide sequences with structural similarity greater than 0.85 to hypoglycemic peptides

[0075]

[0076]

[0077]

[0078]

[0079] Based on the results of the above structural screening, the peptides IPVIA, IPVA, IPTA, IPVVA, IPVPA, VVPL, VPKPL, VPSL, IPPA, VIPL, VPAL, VPVVL, VPQ, VPIIL, HAYVFGVEDTLNALESGAIETLIVWENLDINR and YVFGVEDTLNALESGAIETLIVWENLDINR were screened and synthesized, and their in vitro DPP-IV inhibitory activity, inhibition type, DPP-IV inhibitory activity at the intestinal organoid level and the ability to increase the concentrations of GLP-1 and PYY in the system were measured and analyzed.

[0080] Example 3 Determination of in vitro inhibitory activity and inhibitory type of potential DPP IV inhibitory peptides in the peptide library

[0081] a. Peptide inhibition IC of DPP IV 50 Determination

[0082] The detection was carried out by a chromogenic substrate method using glycylproline p-nitroanilide (Gly-Pro-PNA) as a substrate.

[0083] (1) Preparation of reaction reagents:

[0084] Samples: The 16 polypeptides synthesized after comprehensive screening in Example 2 were dissolved in a PBS solution at pH = 7.4 to prepare six sample solutions with concentration gradients of 5.0, 2.0, 1.0, 0.5, 0.2 and 0.1 mg / mL.

[0085] Substrate: Gly-Pro-pNA was dissolved in Tris-HCl buffer (containing 1.0 mM EDTA) at pH 8.0 to prepare a Gly-Pro-pNA solution with a concentration of 2.0 mM.

[0086] Enzyme: rhDPP-IV was dissolved in 100 mM Tris-HCl (containing 1.0 mM EDTA) at pH 8.0 to prepare a rhDPP-IV solution with a concentration of 0.0625 units / mL.

[0087] Negative control: PBS solution at pH = 7.4.

[0088] (2) Experimental steps:

[0089] Construct the following 100 μL reaction system in a 96-well transparent microplate reader:

[0090] ① Add 60 μL of peptide sample and 20 μL of Gly-Pro-pNA solution and mix well using a plate shaker for 1 minute;

[0091] ② Add 20 μL of rhDPP-IV solution and add Tris-HCl buffer to a total volume of 100 μL, so that the final rhDPP-IV activity is 0.025 Unit / mL and the final concentration of Gly-Pro-pNA is 0.40 mM;

[0092] ③ Place in a microplate reader and incubate at 37°C for 60 minutes, and measure the absorbance at 405nm.

[0093] (3) Analytical methods:

[0094] For any sample, two points where the absorbance value changes within the linear range are selected to calculate the rate of change of absorbance over time S = (Abs2-Abs1) / (t2-t1), and the DPP-IV inhibition rate is calculated by the following formula:

[0095]

[0096] In the formula, IR sample Represents the inhibition rate of the sample on rhDPP-IV, S NC represents the rate of change (slope) of absorbance of the negative control, i.e., the solvent group, in the detection system, S sample Represents the rate of change of absorbance of the sample group. All samples and control groups need to be tested in triplicate and the standard deviation is calculated.

[0097] The peptide concentration was used as the independent variable and the DPP-IV inhibition rate was used as the dependent variable to draw a graph and fit the curve. The concentration corresponding to the DPP-IV inhibition rate of 0.5 was calculated according to the fitting formula, and this concentration was the estimated half-inhibitory concentration IC 50 , the experimental results are shown in Table 5.

[0098] Table 5 Screening peptide IC 50 determination results

[0099]

[0100] (4) Experimental results analysis: From the experimental results, it can be seen that the IC values ​​of 16 peptide samples are 50 The values ​​are all in the mM range, and the IC values ​​of 14 peptides are 50 The value was lower than 0.5mM, and the IC values ​​of 9 peptides 50 The value was lower than 0.1 mM, and the IC 50 The value is 8 nM, among which IPI and VPL (two recognized DPP-IV inhibitory peptide sequences) have an IC 50The values ​​are 0.047 and 0.114 mM, respectively, indicating that the polypeptide sequences IPVIA, IPVA, IPTA, IPVVA, IPVPA, VPKPL, VPSL, IPPA, VPAL, VPVVL, VPQ, VPIIL, VVPL, VIPL, HAYVFGVEDTLNALESGAIETLIVWENLDINR, YVFGVEDTLNALESGAIETLIVWENLDINR, etc. provided in the present application can inhibit DPP-IV more effectively than the polypeptides with potential hypoglycemic effects in existing reports, and are safer and more effective.

[0101] b. Inhibition type determination

[0102] Prepare 100 μM solution of each polypeptide to be tested, and then measure it according to the DPP IV inhibition rate determination method in the above Example 3a., changing the concentration of the chromogenic substrate Gly-Pro-PNA so that its final concentration is 0.1, 0.2, 0.3, 0.4 and 0.5 mM, respectively. At the same time, Tris-Hcl with pH 8.0 is used as a negative control sample. After incubation at 37°C for 60 minutes, the absorbance at 405 nm is recorded with an enzyme reader. Plot the reciprocal of the Gly-Pro-PNA concentration as the independent variable and the reciprocal of the absorbance as the dependent variable, and fit the curve. If the intercepts of the fitting curves of the experimental group and the negative control are the same but the slopes are different, it is competitive inhibition; if the slopes of the fitting curves of the experimental group and the negative control are the same, it is anti-competitive inhibition; if the intersection points of the fitting curves of the experimental group and the negative control with the horizontal axis are the same but the slopes are different, it is non-competitive inhibition. The experimental results are as follows. Figure 2 As shown. Figure 2 It can be seen that all 16 peptides have competitive inhibition.

[0103] Example 4 Evaluation of the Potential Hypoglycemic Activity of Peptides Using an Intestinal Organoid Model

[0104] The 16 peptides obtained in Example 2 were artificially synthesized, and their DPP-IV inhibitory activity, GLP-1 concentration, and PYY concentration measurement experiments were evaluated using a human intestinal organoid model to evaluate their hypoglycemic activity.

[0105] Experimental methods:

[0106] (1) The intestinal organoids induced by human iPSC were inoculated into a 24-well plate and then cultured in an incubator for 72 hours. The culture medium was replaced with a culture medium containing 2 mg / mL of each synthetic peptide and cultured for 48 hours. The supernatant was then taken out and the activity of DPP IV and the concentrations of GLP-1 and PYY in the supernatant were measured. The inhibitory activity of DPP IV was measured using DPPIV-Glo TMProtease Assay (Promega) was used to measure the concentration of GLP-1 and PYY using the ELSA kit of Shanghai Ke Ai Bo Biological. The results are shown in Figure 3-5 shown.

[0107] The analysis results show that all synthetic peptides can inhibit DPP IV activity and promote the secretion of GLP-1 and PYY to a certain extent. Among them, most peptides show high inhibitory activity in inhibiting DPP IV activity, among which the inhibitory effects of peptides IPVIA, IPVA, IPTA, IPVVA, IPVPA, VPKPL, VPSL, IPPA, VPAL, VPVVL, VPQ, and VPIIL are all above 90%; in promoting the secretion of GLP-1, the peptides IPVA, IPVIA, VVPL, VIPL, and VPVVL have the best effects; in promoting the secretion of PYY, the peptides VVPL, IPVA, VIPL, IPVPA, and VPVVL have the best effects.

[0108] The above is only a preferred embodiment of the present application and does not limit the present application in any form. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the content of the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A polypeptide, characterized in that Its amino acid sequence is: any one selected from IPVIA (Seq ID No.1), IPVA (Seq ID No.2), IPTA (Seq ID No.3), IPVVA (Seq ID No.4), IPVPA (Seq ID No.5), VVPL (Seq ID No.6), VPKPL (Seq ID No.7), VPSL (Seq ID No.8), IPPA (Seq ID No.9), VIPL (Seq ID No.10), VPAL (Seq ID No.11), VPVVL (Seq ID No.12), VPIIL (Seq ID No.13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No.15) or VPQ.

2. A nucleic acid sequence encoding the polypeptide of claim 1.

3. A composition comprising one or more polypeptides having an amino acid sequence of IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15) or VPQ.

4. The composition according to claim 3, characterized in that The composition further comprises a pharmaceutically or food-acceptable carrier, auxiliary material or a second active ingredient.

5. The composition according to claim 4, characterized in that The second active ingredient is selected from one or more of a DPP-IV inhibitor, a SGLT-2 inhibitor, a GLP-1 receptor agonist, an insulin secretagogue, an α-glucosidase inhibitor, an angiotensin converting enzyme inhibitor, an angiotensin receptor antagonist, a calcium channel blocker or a β-receptor blocker.

6. Use of the polypeptide according to claim 1 or the composition according to any one of claims 3 to 5 in the preparation of drugs or special medical purpose foods for preventing, treating or improving hyperglycemia and human-related diseases caused by hyperglycemia.

7. A method for preventing, treating or improving hyperglycemia and human-related diseases caused by hyperglycemia, the method comprising administering to a subject an effective amount of a polypeptide, wherein the amino acid sequence of the polypeptide is selected from any one of IPVIA (Seq ID No. 1), IPVA (Seq ID No. 2), IPTA (Seq ID No. 3), IPVVA (Seq ID No. 4), IPVPA (Seq ID No. 5), VVPL (Seq ID No. 6), VPKPL (Seq ID No. 7), VPSL (Seq ID No. 8), IPPA (Seq ID No. 9), VIPL (Seq ID No. 10), VPAL (Seq ID No. 11), VPVVL (Seq ID No. 12), VPIIL (Seq ID No. 13), HAYVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 14), YVFGVEDTLNALESGAIETLIVWENLDINR (Seq ID No. 15) or VPQ.

8. The polypeptide according to claim 1, the composition according to any one of claims 3 to 5, the use according to claim 6, or the method according to claim 7, characterized in that: The polypeptide is extracted from hemp seed.

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