A polypeptide derived from sesame protein and having blood pressure lowering and blood sugar lowering activities

Abstract

The present invention belongs to the technical field of deep processing of agricultural products, and specifically relates to a sesame active polypeptide, which is composed of 9 amino acids, and its sequence is: Ala-Phe-Pro-Ala-Gly-Ala-Ala-His-Trp (AFPAGAAHW). The sesame polypeptide of the present invention can be obtained by enzymatic separation and purification of sesame protein, and can also be artificially synthesized by chemical solid phase synthesis. By measuring ACE inhibition activity and DPP-IV inhibition activity, it is found that the polypeptide shows hypotensive and hypoglycemic activity, and can be used as a functional ingredient in the preparation of drugs, health products, foods, daily chemical products and other fields, and has broad market prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of plant proteins, and specifically relates to a polypeptide AFPAGAAHW derived from sesame 11S globulin isoform 4 (11S globulin isoform 4, EGDILAFPAGAAHWAYNNG 144-152), a preparation method and application of the polypeptide in preparing blood pressure lowering and blood sugar lowering drugs. Background Art

[0002] At present, hypertension and diabetes have become one of the common chronic diseases in my country, and are showing an increasing trend. The complications of the above two diseases are serious and difficult to cure. In addition to bringing a heavy economic burden to the national public health and patients' families, they also seriously affect the quality of life of patients. At present, in order to treat hypertension and diabetes, humans have developed a variety of chemical drugs. Although these drugs are fast-acting, they break the interactive symbiosis between hypertension and diabetes. Patients with hypertension and diabetes need to take multiple drugs at the same time to control the two diseases. In addition, the side effects of taking chemical drugs also endanger human health.

[0003] In order to safely and effectively control hypertension and diabetes, researchers have discovered peptides from food-derived protein hydrolysates that have both antihypertensive (ACE inhibition) and blood sugar (DPP-IV inhibition) activities. Compared with traditional drugs, this type of peptide has the advantages of high safety and antihypertensive and blood sugar lowering activities, and can also be absorbed and utilized by the human body to play a nutritional function. Therefore, it is considered to be a beneficial ingredient for assisting the treatment of hypertension and diabetes, and has broad market prospects and development value.

[0004] Sesame is one of the world's major oil crops. my country is a major producer and consumer of sesame, with an annual production and consumption of about 400,000 tons and 1.5 million tons, respectively. Sesame protein is one of the main components of sesame, accounting for about 20% of sesame. Limited by processing technology, the utilization rate of sesame protein is low. It is currently mainly used as feed and fertilizer, and the product has low added value. Studies have shown that sesame protein can produce antioxidant peptides, blood pressure-lowering peptides, and chelated metal peptides after enzymatic hydrolysis. At the same time, researchers speculated based on the sesame protein sequence that sesame protein can produce DPP-IV inhibitory peptides after appropriate enzymatic hydrolysis. Therefore, it is worth developing and utilizing sesame protein to produce high-value-added active peptides.

[0005] Considering the important social significance of controlling hypertension and diabetes, the broad prospects of the antihypertensive and hypoglycemic markets (the market growth rate of diabetes treatment drugs in my country will be about 7% from 2020 to 2030, reaching 116.7 billion yuan in 2030; the market size of antihypertensive drugs in my country reached 66.31 billion yuan in 2018 and is still growing), and the quality and efficiency improvement of sesame, it is necessary to develop sesame protein-derived peptides that have both the ability to lower blood pressure and blood sugar. Summary of the invention

[0006] In order to meet the needs of people's healthy life, prevent and assist in the treatment of hypertension and diabetes, the purpose of the present invention is to provide a polypeptide derived from sesame protein that has both blood pressure lowering and blood sugar lowering activities. The sesame polypeptide has the advantages of high activity, mature preparation technology, and controllable cost, and has broad market prospects.

[0007] The present invention also provides a method for preparing the sesame polypeptide, and its application in preparing drugs having the effects of lowering blood pressure and / or lowering blood sugar.

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

[0009] A sesame protein-derived polypeptide with blood pressure and blood sugar lowering activity, the polypeptide is composed of 9 amino acids, and its sequence is: Ala-Phe-Pro-Ala-Gly-Ala-Ala-His-Trp (AFPAGAAHW, AW-9), derived from sesame 11S globulin isoform 4 (11S globulin isoform 4, Q2XSW6_SESIN). Specifically, AFPAGAAHW is derived from 144-152 of the protein, namely EGDIL AFPAGAAHW AYNNG 144-152.

[0010] The present invention provides a method for preparing the above polypeptide, which can artificially prepare the polypeptide using conventional solid phase synthesis methods in the art.

[0011] The present invention also provides a method for extracting the above polypeptide from sesame protein, which comprises the steps of protein extraction, proteolysis, separation and purification, and specifically comprises the following steps:

[0012] 1) Extract sesame protein:

[0013] After the sesame seeds are cleaned, they are crushed, defatted with petroleum ether, and the residual solvent is removed. Distilled water is added at a ratio of 1:8-1:20 (w / v, g / ml), the pH is adjusted to 9-11 (to promote protein dissolution), stirred at room temperature for 1-3 hours, centrifuged at 4000-5000 r / min for 20-40 minutes, the supernatant is collected, the pH is adjusted to 4-5 (to precipitate the protein), allowed to stand for 3-6 hours, centrifuged at 4000-5000 r / min for 20-40 minutes, the precipitate is collected, dialyzed, and freeze-dried to obtain sesame protein;

[0014] 2) Proteolysis:

[0015] The sesame protein obtained in step 1) was added to distilled water at a ratio of 1 g: 10-25 mL (w / v) to obtain a sesame protein solution, heated at 100°C for 20-30 min, homogenized at 12000-20000 r / min for 5-10 min while hot, cooled to room temperature, adjusted the pH value to 1.5-2.5, added mixed protease, stirred and hydrolyzed at 30-50°C for 3 h, then adjusted the pH value to 7.5-9.0, added chymotrypsin, ultrasonically assisted hydrolysis at 30-50°C for 2 h, adjusted the pH value to 4.0-4.5, centrifuged at 8000-10000 r / min for 15-30 min, took the supernatant, and kept it at 4-10°C for later use; adjusted the pH value with sodium hydroxide and hydrochloric acid solution;

[0016] The mixed protease is composed of pepsin and acid protease with an enzyme activity ratio of 4:1-7:1;

[0017] When ultrasound-assisted enzymatic hydrolysis is used, the frequency of ultrasound is 20 kHz or 45 kHz, the ultrasound mode is energy-concentrating, and the action mode is intermittent (the action time and rest time are 1:1);

[0018] 3) Separation and purification:

[0019] The pH of the supernatant obtained in step 2) was adjusted to 7.0, separated by ultrafiltration, desalted by nanofiltration, concentrated, and then separated and purified by dextran gel column chromatography. The components with a retention time of 230-325 min were collected and freeze-dried. The peptide sample obtained by freeze-drying was analyzed and identified by Nano-LC-ESI-MS / MS. The amino acid sequence of the peptide with antihypertensive and hypoglycemic activity was identified as: Ala-Phe-Pro-Ala-Gly-Ala-Ala-His-Trp (AFPAGAAHW, AW-9). AFPAGAAHW is derived from sesame 11S protein isomer 4 (Q2XSW6_SESIN) 144-152, that is, EGDIL AFPAGAAHW AYNNG144-152.

[0020] Specifically, in step 2), the 100° C. heating is performed using an ohmic heating method, the electrodes are rectangular, and the power source is alternating current.

[0021] In order to improve the enzymatic hydrolysis effect and achieve sufficient enzymatic hydrolysis of the raw materials, it is further preferred that in step 2), the mixed protease enzymatic hydrolysis and the chymosin enzymatic hydrolysis are both secondary continuous enzymatic hydrolysis, that is, after adding 1% (w / w) sesame protein mass of mixed protease for enzymatic hydrolysis for 1.5h, 1% of sesame mass of mixed protease is added for enzymatic hydrolysis for 1.5h again; after adding 1% (w / w) sesame protein mass of chymosin for enzymatic hydrolysis for 1h, 1% of sesame protein mass of chymosin is added for enzymatic hydrolysis for 1h.

[0022] Furthermore, in step 2), the trypsin is composed of trypsin and chymotrypsin with an enzyme activity ratio of 5:1-9:1.

[0023] Specifically, in step 3), the ultrafiltration is a two-stage ultrafiltration, the molecular weight cutoff of the first-stage ultrafiltration membrane is 10000 Da, the permeate of the first-stage ultrafiltration membrane is separated by a second-stage ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the permeate of the second-stage ultrafiltration is desalted and concentrated by a nanofiltration membrane with a molecular weight cutoff of 500 Da. The above membranes can all be flat membranes or rolled membranes.

[0024] Furthermore, in step 3), the separation and purification conditions of the dextran gel column chromatography were as follows: the column was 60 × 2.6 cm, the mobile phase was distilled water, the flow rate was 1.0 mL / min, the detection wavelength was 220 nm, the sample volume was 4 mL, and P4 was collected (time 230-325 min).

[0025] More preferably, in step 3), dextran LH-20 with a particle size of 40-120 µm is used.

[0026] The present invention also provides the use of the above polypeptide as a main functional component in the preparation of blood pressure lowering and / or blood sugar lowering drugs, health products, foods, daily chemical products, etc.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1) The raw materials used in the present invention are sesame or low-temperature sesame cake, which are widely available, have cost advantages and do not affect the utilization of other sesame components. At the same time, the product has high added value and is worth developing and utilizing;

[0029] 2) The secondary continuous enzymatic hydrolysis has better enzymatic hydrolysis effect, sufficient protein degradation and high yield of target peptide;

[0030] 3) The sesame active polypeptide provided by the present invention has high safety (see Table 2) and has the efficacy of lowering blood pressure and / or blood sugar;

[0031] 4) The sesame polypeptide provided by the present invention has significant activity and can be used not only as a food additive but also as a functional ingredient in medicines, health products and daily chemical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the spectrum of the dextran LH-20 column chromatography of Example 1;

[0033] Figure 2 This is the HPLC chart of the artificially synthesized active polypeptide in Example 2;

[0034] Figure 3is the mass spectrum of the artificially synthesized active polypeptide in Example 2;

[0035] Figure 4 This is the docking diagram of sesame peptide AFPAGAAHW and human angiotensinogenase (1UZE);

[0036] Figure 5 This is the docking diagram of sesame peptide AFPAGAAHW and human DPP-IV (1RWQ). DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the embodiments and drawings, but the protection scope of the present invention is not limited thereto. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in the field without creative work are all within the protection scope of the present invention.

[0038] In the following examples, unless otherwise specified, all raw materials used are common commercial products that can be directly purchased. For example, the acid protease used is a liquid food-grade acid protease purchased from Xiasheng (Shanghai) Biotechnology Co., Ltd., with an enzyme activity of 150,000 U / g.

[0039] Example 1

[0040] A method for extracting sesame polypeptide from sesame protein, which specifically comprises the following steps:

[0041] 1) Sesame protein extraction:

[0042] Take 1kg of sesame without impurities and mold, crush it to 40 mesh, and use a Soxhlet extractor for degreasing. The degreasing conditions are: petroleum ether (boiling range 30-60℃) degreasing at 65℃ for 10 hours. Add distilled water to the defatted sesame powder at a material-liquid ratio (w / v) of 1 g:20 mL, adjust the pH to 10 with NaOH, stir at room temperature for 120 minutes for protein extraction, then centrifuge at 4500 r / min for 30 minutes, take the supernatant, adjust the pH to 4.3, let it stand for 3 hours, centrifuge at 4500 r / min for 30 minutes, collect the precipitate, and dialyze it for 12 hours using a dialysis bag (molecular weight cutoff 3000 Da). The distilled water is replaced 6 times during the dialysis. After dialysis, the liquid is freeze-dried at -60℃ to obtain sesame protein, which is sealed for later use.

[0043] 2) Proteolysis:

[0044] The sesame protein obtained in step 1) was added to distilled water at a material-liquid ratio of 1 g:10 mL (w / v) to obtain a sesame protein solution, which was heated at 100°C for 25 min in an ohmic heating tank, homogenized at 15000 r / min for 5 min while hot, cooled to room temperature, adjusted to pH 2.0, added with a 1.0% mixed protease (the mixed protease consisted of pepsin and acid protease with an enzyme activity ratio of 7:1) of sesame protein, stirred at 37°C for 1.5 h, and then added with a 1.0% mixed protease for 1.5 h. Subsequently, the pH was adjusted to 8.0, and 1.0% sesame protein chymotrypsin (the chymotrypsin consisted of trypsin and chymotrypsin with an enzyme activity ratio of 6:1) was added to the solution, and then 1.0% sesame protein chymotrypsin was added to the solution for 1 h at 37°C, and then 1.0% sesame protein chymotrypsin was added to the solution for 1 h. During the enzymatic hydrolysis of pancreatic chymotrypsin, ultrasound was used to assist the enzymatic hydrolysis, and the ultrasonic conditions were: frequency 45 kHz, ultrasonic mode was energy-concentrating type, and the action mode was intermittent (action time and rest time were 1:1). After the enzymatic hydrolysis was completed, the pH was adjusted to 4.3, centrifuged at 10,000 r / min for 30 min, and the supernatant was collected and stored at 4-10°C for later use.

[0045] 3) Separation and purification

[0046] The supernatant obtained in step 2) is adjusted to pH 7.0 and separated by two-stage ultrafiltration. The conditions of the first-stage ultrafiltration are: operating pressure 1.5 MPa, and the ultrafiltration membrane is 10000 Da. The permeate of the first-stage ultrafiltration is then treated by secondary ultrafiltration. The operating pressure of the secondary ultrafiltration is 1.5 MPa, and the ultrafiltration membrane is 3000 Da. The permeate of the secondary ultrafiltration is desalted and concentrated by nanofiltration (the molecular weight cutoff of the nanofiltration membrane is 500 Da). After concentrating to 10% of the original feed volume, distilled water is added to restore it to the original volume, and then nanofiltration is concentrated and desalted again. The above operation is repeated three times, and then the nanofiltration retentate is collected. The above-mentioned ultrafiltration membrane and nanofiltration membrane are both roll membranes.

[0047] Take 4 mL of the nanofiltration retentate and load it onto a dextran LH-20 column (60 × 2.6 cm) at a flow rate of 1.0 mL / min. The mobile phase was distilled water. The detection wavelength was 220 nm. The peak P4 component with a retention time of 230-325 min was collected (see Figure 1 ), freeze-dried at -60℃ for 24-48 h to obtain sesame peptide products.

[0048] The peptide samples were analyzed and identified by Nano-LC-ESI-MS / MS, and the amino acid sequence of the peptide with antihypertensive and hypoglycemic activities was identified as: Ala-Phe-Pro-Ala-Gly-Ala-Ala-His-Trp (AFPAGAAHW).

[0049] Example 2

[0050] A method for artificially synthesizing sesame polypeptides, which is synthesized artificially by solid phase synthesis. The basic process is as follows: first, an amino acid whose amino group is protected by an Fm℃ group is connected to an insoluble solid phase carrier Wang resin, and then the amino group's protecting group is removed, and the first amino acid is connected to the solid phase carrier; secondly, the carboxyl group of the second amino acid whose amino group is protected by an Fm℃ group is activated with a condensing agent, and the activated amino acid reacts with the amino group of the first amino acid connected to the solid phase carrier to form a peptide bond, and at this time, a dipeptide with a protecting group is generated on the solid phase carrier. Repeat the above-mentioned peptide bond formation reaction to allow the peptide chain to grow from the C-terminus to the N-terminus until the required peptide chain length is reached, and finally cut to obtain the target polypeptide Ala-Phe-Pro-Ala-Gly-Ala-Ala-His-Trp (AFPAGAAHW). The liquid phase mass spectrometry analysis diagram of the synthesized sesame polypeptide sequence is shown in Figure 2 and Figure 3 The mass-to-charge ratio of the main ion peak of this high-purity synthetic peptide is 927.80, which is consistent with the molecular weight of the required synthetic sequence, indicating that the solid phase synthesis is successful.

[0051] The specific process of synthesizing sesame polypeptides by solid phase synthesis method is as follows: ① Huang Weide and Chen Changqing, Peptide Synthesis, Science Press, 1985. ② N. Heward, HD Jakubko, translated by Liu Keliang et al., Peptides: Chemistry and Biology, Science Press, 2005. ), the sesame polypeptide can also be commissioned to the corresponding polypeptide biological company for synthesis, because its chemical synthesis process is not the focus of this application, so this application will not describe the chemical synthesis process in detail here.

[0052] Application test of antihypertensive and hypoglycemic activity

[0053] In the following application test, the determination of ACE inhibition ability (lowering blood pressure) and DPP-IV inhibition ability (lowering blood sugar) is carried out as follows. The polypeptide solution is prepared by adding lyophilized or artificially synthesized sesame polypeptide to dilute it with distilled water to prepare an aqueous solution of a certain concentration.

[0054] 1) ACE inhibition ability determination:

[0055] Take 20 μL of 1 mg / mL AFPAGAAHW peptide solution, mix it with 40 μL of angiotensin converting enzyme (ACE) (12.5mμ / mL) and 40 μL of 4.66 mmol / L hippuryl-histidyl-leucine (HHL) solution (0.4 mmol / L pH8.5 phosphate buffer containing 0.6 mol / L sodium chloride), react at 37℃ for 1 h, add 150 μL of 1.2 mol / L NaOH to terminate the reaction, then add 40 μL of 2% o-phthalaldehyde (OPA) methanol solution to react at room temperature for 20 min, and then add 40 mL of 6mol / L HCl to terminate the reaction. After the reaction solution is diluted with distilled water at a ratio of 1:15, pour it into a fluorescence cuvette, set the excitation wavelength to 340 nm, the emission wavelength to 455 nm, and the slit width to 5 nm, and measure the fluorescence intensity. Then calculate according to the following formula:

[0056]

[0057] Wherein: a is the fluorescence intensity of the solution containing AFPAGAAHW, ACE and HHL, b is the fluorescence intensity of the solution containing ACE and HHL, c is the fluorescence intensity of the solution containing peptide and HHL, and d is the fluorescence intensity of the solution containing HHL.

[0058] 2) Determination of DPP-IV inhibitory ability:

[0059] Take glycylproline p-nitroanilide and add it to 100 mmol / L Tris-HCl buffer at pH8 to make a 1.6 mmol / L solution. Take 25 μL and add it to a 96-well plate. Add 25 μL of 1 mg / mL AFPAGAAHW solution (the solvent is 100 mmol / L Tris-HCl buffer at pH8) or preheat at 37°C for 10 min, add 50 μL of 8 U / L dipeptidyl peptidase IV (DPP-IV), mix well, react at 37°C for 60 min, add 100 μL of 1 mol / L pH4.0 acetate buffer to terminate the enzyme reaction, and use a microplate reader to measure the absorbance at 405 nm. In the control group experiment, 25 μL of 100 mmol / L pH8 Tris-HCl was used instead of AFPAGAAHW solution, and other conditions were the same. Calculate the inhibition rate according to the following formula:

[0060]

[0061] Where A S is the absorbance in the presence of AFPAGAAHW, A CThe absorbance of AFPAGAAHW was replaced by an equal volume of Tris-HCl buffer. SE The absorbance of DPP-IV was replaced by an equal volume of Tris-HCl buffer. CE The absorbance of DPP-IV and HPSPR was replaced by Tris-HCl.

[0062] The active application of sesame polypeptides, ACE inhibition rate as a blood pressure lowering index, DPP-IV inhibition rate as a blood sugar lowering index, the test analysis of the blood pressure lowering and blood sugar lowering activities of the sesame polypeptide extracted from sesame protein in Example 1 and the artificially synthesized sesame polypeptide AFPAGAAHW in Example 2 are basically the same, both showing strong blood pressure lowering and blood sugar lowering abilities (see Table 1).

[0063] Table 1. Antihypertensive and hypoglycemic activities of sesame polypeptide AFPAGAAHW obtained in Examples 1 and 2

[0064]

[0065] Note: When measuring ACE inhibitory activity, the concentration of AFPAGAAHW solution is 0.06 mg / mL. When measuring DPP-IV inhibition rate, the concentration of AFPAGAAHW solution is 0.125 mg / mL.

[0066] Table 2 Physiological properties of sesame polypeptide AFPAGAAHW obtained in Examples 1 and 2

[0067]

[0068] Note: The numbers in brackets are prediction probabilities, and physiological characteristics were predicted using admetSAR (http: / / lmmd.ecust.edu.cn / admetsar2 / ).

[0069] It can be seen from Table 2 that AFPAGAAHW is highly safe, predicted to be non-toxic and non-carcinogenic, and will not affect the normal physiological functions of the human body. Figure 4 It can be seen that AFPAGAAHW affects Glu384, Tyr523, Gln281, Lys511, His353, His513, Zn 2+ 701, His383 active site, thereby changing the spatial position of ACE, affecting its binding with angiotensin and playing a role in lowering blood pressure. Figure 5 It can be seen that AFPAGAAHW interacts with the active sites of Glu205, Arg125, and Tyr631 in DPP-IV through hydrogen bonds and hydrophobic interactions, affecting the function of DPP-IV and playing a hypoglycemic role.

[0070] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Use of a polypeptide in the preparation of a hypoglycemic drug, characterized in that: The polypeptide consists of 9 amino acids, and its sequence is: Ala-Phe-Pro-Ala-Gly-Ala-Ala-His-Trp.

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