Fagopyrum tataricum protein source blood fat reducing peptide and application thereof

Abstract

The invention relates to a tartary buckwheat protein source blood fat reducing peptide and application thereof. The sequence of the tartary buckwheat protein source hypolipidemic peptide is one or more of the following sequences: FHWDYPQA; fHWDYPQALE is used as a template; and LFHWDYPQA is adopted. The tartary buckwheat protein source hypolipidemic peptide has excellent pancreatic lipase inhibition capability, can effectively inhibit differentiation of preadipocytes, and has the capabilities of reducing total cholesterol, reducing triglyceride, reducing density lipoprotein cholesterol and improving high density lipoprotein cholesterol. In addition, the tartary buckwheat protein source hypolipidemic peptide is non-toxic and high in safety.

Description

Technical Field

[0001] The present invention relates to the field of small molecule peptides, and more specifically, to a tartary buckwheat protein-derived lipid-lowering peptide and application thereof. Background Art

[0002] Obesity is a metabolic disease caused by long-term intake of a high-sugar and high-fat diet. It is closely related to other chronic diseases, such as type 2 diabetes, fatty liver, and cardiovascular disease. Obesity may lead to pathological changes such as increased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and decreased high-density lipoprotein cholesterol (HDL-C), and may further lead to a variety of diseases or complications such as hyperlipidemia. Therefore, the development of new food-derived lipid-lowering peptides is of great significance to supplement and improve existing obesity management strategies.

[0003] Bioactive peptides are protein fragments that have a positive effect on body functions and can regulate and affect physical health. Many bioactive peptides from animal and plant sources have good lipid-lowering activity. Tartary buckwheat is widely used for its nutritional components and beneficial effects on health, especially for the treatment of chronic diseases such as diabetes, obesity, and cardiovascular disease. For example, Ma et al. (DOI: 10.1016 / j.foodchem.2005.01.052) isolated peptides with angiotensin I converting enzyme activity from common buckwheat, but there are still few studies on the active peptides of tartary buckwheat protein.

[0004] Therefore, it is of great significance to develop more active buckwheat protein peptides with lipid-lowering activity. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the problem that the existing active peptides of tartary buckwheat protein with lipid-lowering activity are still relatively lacking, and to provide a lipid-lowering peptide derived from tartary buckwheat protein. The lipid-lowering peptide derived from tartary buckwheat protein has excellent pancreatic lipase inhibition ability, can effectively inhibit the differentiation of preadipocytes, and has the ability to reduce total cholesterol, triglycerides, low-density lipoprotein cholesterol and increase high-density lipoprotein cholesterol. In addition, the lipid-lowering peptide derived from tartary buckwheat protein of the present invention is non-toxic and highly safe.

[0006] A further object of the present invention is to provide the use of the above-mentioned tartary buckwheat protein-derived lipid-lowering peptide in the preparation of a drug for preventing and / or treating diseases related to hyperlipidemia.

[0007] Another object of the present invention is to provide the use of the above-mentioned tartary buckwheat protein-derived lipid-lowering peptide in the preparation of a health product that assists in lowering blood lipids.

[0008] Another object of the present invention is to provide a composition.

[0009] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0010] A tartary buckwheat protein-derived lipid-lowering peptide, wherein the tartary buckwheat protein-derived lipid-lowering peptide is at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0011] The tartary buckwheat protein-derived lipid-lowering peptide of the present invention is extracted from tartary buckwheat, and the sequence of the tartary buckwheat protein-derived lipid-lowering peptide is one or more of the following sequences: FHWDYPQA (phe-his-trp-asp-tyr-pro-gln-ala); FHWDYPQALE (phe-his-trp-asp-tyr-pro-gln-ala-leu-glu); LFHWDYPQA (leu-phe-his-trp-asp-tyr-pro-gln-ala).

[0012] The inventors of the present invention have found through research that the bitter buckwheat protein-derived hypolipidemic peptide of the present invention has excellent pancreatic lipase inhibition ability, can effectively inhibit the differentiation of pre-adipocytes, and has the ability to reduce total cholesterol, triglycerides, and low-density lipoprotein cholesterol and increase high-density lipoprotein cholesterol. In addition, the bitter buckwheat protein-derived hypolipidemic peptide of the present invention is non-toxic and highly safe.

[0013] The tartary buckwheat protein-derived hypolipidemic peptide of the present invention can be obtained by enzymatic hydrolysis, separation and purification of tartary buckwheat, or by solid phase synthesis. Since it can be obtained by solid phase synthesis, it can be mass-produced, and the synthesized tartary buckwheat protein-derived hypolipidemic peptide has high purity, stable product quality, and market competitiveness.

[0014] Preferably, the tartary buckwheat protein-derived hypolipidemic peptide is a sequence shown in SEQ ID NO: 1. This sequence can more effectively inhibit the differentiation of preadipocytes.

[0015] Preferably, the tartary buckwheat protein-derived lipid-lowering peptide is a sequence shown in SEQ ID NO: 2. This sequence is more conducive to lowering low-density lipoprotein cholesterol.

[0016] Preferably, the tartary buckwheat protein-derived lipid-lowering peptide is the sequence shown in SEQ ID NO: 3. This sequence has a higher inhibition rate on pancreatic lipase, is more conducive to reducing total cholesterol and triglycerides, and is more conducive to increasing high-density lipoprotein cholesterol.

[0017] The use of the above-mentioned tartary buckwheat protein-derived lipid-lowering peptide in the preparation of drugs for preventing and / or treating diseases related to hyperlipidemia is also within the protection scope of the present invention.

[0018] Preferably, the lipid-lowering related disease is obesity and / or hyperlipidemia.

[0019] Preferably, the drug is a drug that inhibits preadipocyte differentiation.

[0020] Preferably, the drug is a drug for lowering total cholesterol.

[0021] Preferably, the drug is a triglyceride-lowering drug.

[0022] Preferably, the drug is a drug for lowering high-density lipoprotein cholesterol.

[0023] Preferably, the drug is a drug for increasing high-density lipoprotein cholesterol.

[0024] Preferably, the drug is a drug that inhibits pancreatic lipase activity.

[0025] Preferably, the dosage form of the drug is at least one of injection, tablet, oral solution, granule or capsule.

[0026] The present invention also protects the use of the tartary buckwheat protein-derived lipid-lowering peptide in the preparation of a health product for assisting in lowering blood lipids.

[0027] A composition comprises the above-mentioned tartary buckwheat protein-derived lipid-lowering peptide.

[0028] Preferably, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The tartary buckwheat protein-derived lipid-lowering peptide of the present invention has excellent pancreatic lipase inhibition ability, can effectively inhibit the differentiation of pre-adipocytes, and has the ability to reduce total cholesterol, triglycerides, low-density lipoprotein cholesterol and increase high-density lipoprotein cholesterol. In addition, the tartary buckwheat protein-derived lipid-lowering peptide of the present invention is non-toxic and highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The pancreatic lipase inhibition ability diagram of two ultrafiltration fractions MW < 3kDa, MW > 3kDa and enzymatic hydrolysate; Figure 1 A is the PL inhibition rate of each component at different concentrations; Figure 1 B is the PL inhibition rate IC of each component 50 .

[0032] Figure 2 It is the elution peak diagram of the MW < 3kDa component separated by RP-HPLC and the pancreatic lipase inhibition ability diagram of the P1 to P9 components; wherein, Figure 2 A is the elution peak diagram of the MW < 3 kDa component separated by RP-HPLC; Figure 2B is a graph showing the pancreatic lipase inhibitory ability of fractions P1 to P9.

[0033] Figure 3 The binding sites and interaction forces between three tartary buckwheat protein-derived lipid-lowering peptides and pancreatic lipase (PDB: 1LPB).

[0034] Figure 4 The binding sites and interaction forces between three tartary buckwheat protein-derived lipid-lowering peptides and cholesterol esterase (PDB: 1F6W).

[0035] Figure 5 The graph shows the pancreatic lipase inhibitory ability of three lipid-lowering peptides derived from buckwheat protein.

[0036] Figure 6 Figure 2 shows the toxicity test results of three buckwheat protein-derived hypolipidemic peptides and orlistat.

[0037] Figure 7 These are the results of Oil Red O staining of three buckwheat protein-derived lipid-lowering peptides and orlistat.

[0038] Figure 8 This is the absorbance value at 520nm of three buckwheat protein-derived hypolipidemic peptides and orlistat after lipid droplets stained with Oil Red O were dissolved in isopropanol.

[0039] Fig. 9 This is a diagram for determining the lipid-lowering activity of buckwheat protein-derived lipid-lowering peptides in 3T3-L1 preadipocytes; Fig. 9 A is the effect diagram on TG content; Fig. 9 B is the effect diagram on TC content; Fig. 9 C is the effect diagram on HDL-C content; Fig. 9 D is the effect diagram on LDL-C content. DETAILED DESCRIPTION

[0040] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0041] The method for determining the pancreatic lipase (PL) inhibition rate of the present invention is as follows:

[0042] 1mg / mL lauric acid-4-nitrophenyl ester solution was used as the reaction substrate, and the reaction substrate contained 0.05mol / L sodium acetate solution and 1% Triton X-100 solution. The reaction substrate was heated to dissolve, mixed thoroughly, and cooled to room temperature for use. In a 2mL test tube, the reaction substrate, the sample solution to be tested, and the reaction buffer (pH8.2, 0.1mol / L Tris-HCl buffer) were added in sequence, and finally the pancreatic lipase solution (5mg / mL) was added to start the reaction. The volume ratio of the sample was 5:2:4:3. After constant temperature incubation at 37°C for 2h, the absorbance was measured at a wavelength of 420nm. At the same time, a sample group without enzyme and a blank group without sample were set up. The pancreatic lipase inhibition rate (PL) was calculated according to the following formula: PL (%) = [1-(A-A1) / A0] × 100;

[0043] Wherein, A is the absorbance value of the sample group to be tested; A1 is the absorbance value of the sample group without enzyme; A0 is the absorbance value of the blank group without sample.

[0044] The test data processing method of each embodiment of the present invention is as follows:

[0045] The obtained data were plotted using Prism 8.0.2 software and Origin 2019, and statistically processed using SPSS21.0 and Excel2021 software. The results were expressed as mean ± standard deviation (SD), and the ANOVA method was used to analyze the significance of the differences between samples (P<0.05). Each experiment was repeated 3 times.

[0046] Example 1 Extraction and enzymolysis of buckwheat protein, and separation and purification of the enzymolyzate

[0047] 1. Extraction and enzymatic hydrolysis of buckwheat protein

[0048] Tartary buckwheat (Xiqiao No. 8, provided by Xichang University) was crushed, passed through a 100-mesh sieve, and degreased for 2 hours with petroleum ether at a solid-liquid ratio of 1:5 (g / mL). After repeated degreasing twice, it was dried in a fume hood to obtain defatted buckwheat powder. Based on the principle of alkali dissolution and acid precipitation, defatted buckwheat powder was mixed with water at a ratio of 1:10 (g / mL), and the pH was adjusted to 9.0 with 0.1 mol / L NaOH. The mixture was stirred and extracted for 2 hours in a constant temperature water bath at 45°C, centrifuged at 4000 r / min for 20 minutes, and the supernatant was taken. The pH of the supernatant was adjusted to 4.4 with 0.1 mol / L HCl, and the protein was precipitated at 4°C for 2 hours. The precipitate was freeze-dried for later use.

[0049] A 3% crude buckwheat protein solution was prepared, and the optimal enzymatic hydrolysis process was referred to the literature "Enzymatic Preparation, Amino Acid Composition and Activity of Buckwheat Protein Lipid-lowering Peptides" (DOI: 10.13386 / j.issn1002-0306.2023110029), and bromelain was used for enzymatic hydrolysis. The enzyme was inactivated at 95°C, and the solution was cooled and centrifuged at 8000r / min for 20min. The supernatant was the enzymatic hydrolyzate containing buckwheat protein-derived lipid-lowering peptides, which was freeze-dried to obtain the enzymatic hydrolyzate.

[0050] 2. Separation and purification of enzymatic hydrolysates

[0051] The hydrolysate was separated by an ultrafiltration membrane with a molecular weight cutoff (MW) of 3 kDa (Merck Millipore, Billerica, MA, USA), and two ultrafiltration fractions were obtained: MW>3 kDa and MW<3 kDa. The two ultrafiltration fractions and the hydrolysate were freeze-dried and prepared into test sample solutions of different concentrations (0.1, 0.25, 0.5, 1, 2, 4 mg / mL), and their pancreatic lipase inhibition rates and IC50 values ​​were determined. The results are shown in Table 1. Figure 1 shown.

[0052] The pancreatic lipase inhibition ability of the two ultrafiltration fractions MW < 3 kDa, MW > 3 kDa and the enzymatic hydrolysate is as follows Figure 1 As shown. At each mass concentration, the pancreatic lipase inhibition rate of the MW < 3kDa component was significantly higher than that of the hydrolysate component and the MW > 3kDa component. At a concentration of 4 mg / mL, the inhibition rate of the MW < 3KDa component on pancreatic lipase reached 68.65% ± 0.34%. By calculating the IC 50 It can also be seen that the IC of the MW < 3kDa component 50 The lowest was only 0.019 mg / mL. Therefore, the fraction with MW < 3 kDa was selected for further purification.

[0053] The ultrafiltration fraction with MW < 3 kDa was purified by reverse phase high performance liquid chromatography (RP-HPLC). The ultrafiltration fraction with MW < 3 kDa was filtered through a 0.22 μm filter membrane and separated using a C18 reverse phase column (20 mm × 450 mm, 10 μm). A total of 9 fractions were separated and named P1 to P9. The chromatographic conditions were as follows: mobile phase A: double distilled water containing 0.1% trifluoroacetic acid (TFA); mobile phase B: methanol containing 0.1% TFA; elution gradient: 0-40 min, 8% to 50% mobile phase B; 40 to 80 min, 50% to 75% mobile phase B; 80 to 85 min, 75% to 90% mobile phase B; 85 to 95 min, 90% to 90% mobile phase B, injection volume 5 mL, flow rate 10 mL / min, detection wavelengths 214 nm and 280 nm. The eluted peaks were collected, concentrated, freeze-dried and prepared at 1 mg / mL concentrations to determine their pancreatic lipase inhibitory ability and screen out the components with the strongest pancreatic lipase inhibitory rate. The results are as follows Figure 2 shown.

[0054] Figure 2 The figure shows the elution peak of the MW < 3 kDa component separated by RP-HPLC and the pancreatic lipase inhibition ability of the P1 to P9 components. Figure 2 It can be seen that the P9 component has the highest pancreatic lipase inhibitory activity. At a concentration of 1 mg / mL, its pancreatic lipase inhibition rate reached 68.23% ± 0.98%, which is significantly higher than other components. Therefore, we selected the P9 component for the subsequent identification of peptide components.

[0055] Example 2 Identification of peptide composition, molecular docking and determination of pancreatic lipase inhibition rate

[0056] 1. Identification of P9 components by HPLC-MS / MS

[0057] The P9 component was analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was an Orbitrap Q-Exactive Plus mass spectrometer (ThermoFisherScientific, MA, USA) connected in series with an EASY-nanoLC1200. A total of 1 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient of 60 min. The column flow was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 2% of phase B, increased to 35% in 47 min with a nonlinear gradient, increased to 100% within 1 min, and maintained for 12 min.

[0058] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200-1800; resolution: 70,000; AGC target: 3e6; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 17,500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28%; dynamic exclusion time: 30 s.

[0059] Tandem mass spectra were analyzed by PEAKSStudio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKSDB searched the uniprot-Fagopyrum tataricum (version 2023, 382 entries) database and set None digestion. The search parameters were fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, variable modification: Oxidation (M) 15.99, Deamination (NQ) 0.98, protein card value was at least 1 unique peptide; peptide card value was -10lgP ≥ 20.

[0060] A total of 2662 peptide sequences were identified for the P9 component using the above method, of which 40 peptide sequences were oligopeptides with a peptide length of no more than 10 amino acids and no modification groups.

[0061] 2. Molecular docking

[0062] The docking software used was Autodock vina 1.1.2. Pancreatic lipase (PDB ID: 1LPB) and cholesterol esterase (PDB ID: 1F6W) were selected as docking receptors, and their three-dimensional structures were downloaded from the Protein Data Bank (PDB). The selected peptide sequences were mapped using Discovery Studio 2019, and the original ligands methoxyundecylphosphonic acid (MUP) and metal ions of the receptor protein 1LPB were separated and docked. The receptor protein 1F6W did not contain the original ligand and could be docked directly. Next, the protein receptor was dehydrated and hydrogenated, and then the ligand energy was minimized and their rotation centers and rotation bonds were detected. After importing the receptor and ligand into the docking software, the parameters of the docking box were set. The docking parameters of the peptide segment with the active site of 1LPB were set as follows: center coordinates: x = -6.05, y = 28.03, z = 38.479; docking box size: x = 79.2, y = 66.0, z = 77.73, energy_range = 3, exhaustion = 8, number modes = 10. The docking parameters of the peptide segment with the active site of 1F6W were set as follows: center coordinates: x = 3.189, y = 4.989, z = 17.976; docking box size: x = 74.894, y = 60.161, z = 69.983, energy_range = 3, exhaustion = 8, number modes = 10. The docking conformation with the lowest binding energy was selected as the optimal conformation. The three-dimensional structure diagram of the interaction between the peptide segment and the enzyme was drawn using PYMOL, and the visualization was performed using DiscoveryStudio2019. A two-dimensional interaction diagram was drawn to analyze the interaction type after docking.

[0063] The 40 peptides were ranked according to the molecular docking binding energy. The smaller the binding energy, the better the binding effect. A total of three tartary buckwheat protein-derived lipid-lowering peptides were screened, namely FHWDYPQA, FHWDYPQALE and LFHWDYPQA. They will be named using the first and last letters of the peptide sequence and the length of the peptide, namely FA-8, FE-10 and LA-9. The identification results of tartary buckwheat protein-derived lipid-lowering peptides and their peptide characteristics are shown in Table 1.

[0064] Table 1 Identification results and peptide characteristics of tartary buckwheat protein-derived lipid-lowering peptides

[0065]

[0066] Figure 3 The binding sites and interaction forces between three tartary buckwheat protein-derived lipid-lowering peptides and pancreatic lipase (PDB: 1LPB). Figure 3It can be seen that FA-8 forms a salt bridge with 1LPB at amino acid residue ASP328, generates electrostatic interaction with amino acid residue ASP12, and forms π-anionic electrostatic interaction with amino acid residues ARG337 and LYS367. It forms hydrogen bonds with amino acid residues SER47, ARG44, ASP389, ARG337, ASP387, ASN229, ASP328, LYS367, ASP331, and GLU13. It forms a carbon-hydrogen bond with amino acid residue ALA43. It forms an amide-π stacking hydrophobic interaction with amino acid residue GLY236, and forms π-alkyl interactions with amino acid residues ALA332, ALA40, LYS239, and PRO235.

[0067] FE-10 formed salt bridges and electrostatic interactions with 1LPB at amino acid residues ASP328, GLU370 and ASP387, respectively, hydrogen bonds with amino acid residues GLN29, GLU48, GLY14, CYS39, ARG44, LEU41, CYS61 and LYS367, carbon-hydrogen bonds with amino acid residues ALA43 and GLU48, π-anionic interactions with amino acid residues ARG337 and LYS60, and π-alkyl interactions with amino acid residues ALA332, ALA40, LEU41 and LYS367.

[0068] LA-9 formed salt bridges with 1LPB at amino acid residues ASP387 and ASP331, generated electrostatic interactions with amino acid residue ASP12, formed π-anionic interactions with amino acid residue LYS24, formed hydrogen bonds with amino acid residues GLU64, THR292, ASP331, ASP387, ASN294, ASP328, ARG339, ARG337, LYS367, ALA332, and ASN384, formed π-sigma hydrophobic interactions with amino acid residue LEU41, formed amide-π stacking hydrophobic interactions with amino acid residue GLU13, and formed π-alkyl interactions with amino acid residues ALA40, LYS239, LEU41, and LYS367.

[0069] Figure 4 The binding sites and interaction forces between three tartary buckwheat protein-derived lipid-lowering peptides and cholesterol esterase (PDB: 1F6W). Figure 4It can be seen that FA-8 forms hydrogen bonds and carbon-hydrogen bonds with 1F6W at amino acid residues LEU529, THR531, LYS355, LEU527, LYS231, VAL285, PRO226, and PHE351, and generates π-πT-shape, π-alkyl, and alkyl hydrophobic interactions with amino acid residues HIS283, LEU282, PRO226, VAL391, LEU527, and ILE301.

[0070] FE-10 formed hydrogen bonds with 1F6W at amino acid residues ASN232, VAL285, PHE351, LYS231, and PRO226, formed a carbon-hydrogen bond with amino acid residue LYS231, formed π-cationic electrostatic interactions with amino acid residues LYS355 and LYS231, formed π-sigma hydrophobic interactions with amino acid residues HIS283 and ILE301, and formed π-πT-shape, π-alkyl, and alkyl hydrophobic interactions with amino acid residues TRP522, ILE353, LYS231, PRO396, VAL395, VAL391, PRO226, LEU224, ILE301, LEU527, ILE353, TRP236, and PHE235, respectively.

[0071] LA-9 and 1F6W formed hydrogen bonds and carbon-hydrogen bonds at amino acid residues ILE229, LYS231, and PRO226, respectively, generated electrostatic interactions at amino acid residue ASP299, π-anionic electrostatic interactions at amino acid residue LYS231, and π-alkyl hydrophobic interactions at amino acid residues PRO226, ILE229, PRO300, ILE301, LEU224, ILE399, LEU282, and ILE353.

[0072] The above results indicate that the three lipid-lowering peptides derived from buckwheat protein bind to receptors through hydrogen bonds, π-π bonds, hydrophobic, electrostatic and other interactions. These interactions enhance the binding of peptides to receptor proteins or change the spatial conformation of enzymes, thereby affecting their normal physiological functions and showing a lipid-lowering effect.

[0073] 3. Determination of pancreatic lipase inhibition rate

[0074] Pancreatic lipase (PL) is one of the key targets for weight loss and is responsible for digesting nearly 70% of dietary fat intake. The three tartary buckwheat protein-derived lipid-lowering peptides FA-8, FE-10, and LA-9 obtained after molecular docking were prepared into sample solutions of different concentrations (50, 100, 200, 400, and 800 μg / mL), and Orlistat, a key drug for the treatment of obesity, was selected as a positive control to determine the inhibition rate of pancreatic lipase. The results are as follows: Figure 5 shown.

[0075] from Figure 5 It can be seen that when the concentration of buckwheat protein-derived lipid-lowering peptides is 50-800 μg / mL, they have a strong inhibitory effect on pancreatic lipase and are concentration-dependent. Among them, when the concentration is 800 μg / mL, LA-9 (LFHWDYPQA) has the highest inhibition rate of 83.17% ± 0.52% on pancreatic lipase; FA-8 and FE-10 are second, with inhibition rates of 78.52% ± 0.29% and 75.59% ± 0.55% on pancreatic lipase, respectively. The inhibition rates of the three buckwheat protein-derived lipid-lowering peptides on pancreatic lipase are higher than those of existing bioactive peptides on pancreatic lipase, such as the inhibition rate of red bean protein hydrolysate IFNNDPNNHP on pancreatic lipase is 62.60% at 4 mg / mL (DOI: 10.1016 / j.foodchem.2023.138129). This indicates that the tartary buckwheat protein-derived lipid-lowering peptide of the present invention has excellent pancreatic lipase inhibitory ability.

[0076] Example 3 Toxicity determination of tartary buckwheat protein-derived hypolipidemic peptides

[0077] FHWDYPQA, FHWDYPQALE and LFHWDYPQA were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. (Nanjing, Jiangsu, China). The purity of the obtained peptide monomer was 98%, and its toxicity and hypolipidemic activity were subsequently studied.

[0078] 3T3-L1 preadipocytes were cultured in a high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin, i.e., complete medium, and placed in a cell culture incubator at 5% CO2 and 37°C. 100 μL of cell suspension (1×10 5 Cells / mL) were inoculated in a 96-well plate, and a blank group and a test group were set up, wherein the test group included FA-8, FE-10, LA-9 and orlistat. After 24 hours of cell culture, the culture medium was discarded, 100 μL of culture medium was added to the blank group, and 100 μL of culture medium containing samples of different concentrations was added to the test group. After 24 hours of continued culture, the old culture medium was discarded, 100 μL of MTT (0.5 mg / mL) was added and incubated for 4 hours, 100 μL of DMSO was added to each well, and the absorbance was detected at 490 nm. Cell viability was calculated by the following formula: Cellviability(%)=(A t / A c )×100%; where A t is the absorbance value of the test group; A c is the absorbance value of the blank group.

[0079] Figure 6This is the toxicity test result of buckwheat protein-derived hypolipidemic peptide and orlistat. Figure 6 It can be seen that when the concentration of buckwheat protein-derived hypolipidemic peptide and orlistat is 1-80 μg / mL, the cell survival rate is above 90%, indicating that they have no cytotoxicity within this concentration range and can be used for subsequent experiments.

[0080] Example 4 Determination of the lipid-lowering activity of buckwheat protein-derived lipid-lowering peptides

[0081] 1. Differentiation induction of high-fat model

[0082] 3T3-L1 preadipocytes were seeded in 24-well plates (5 × 10 4 Cells / well) were added and cultured in DMEM medium until the cell density reached 100%. The culture medium was replaced and cultured for two days to allow the cells to exit the growth cycle by contact inhibition. A blank control group, a positive control group, a model group, and a test group were set up. The model group was cultured with primary differentiation medium, i.e., complete medium containing 0.5mM IBMX, 1μM DEX, and 10μg / mL insulin for 3 days. The test group was cultured with primary differentiation medium containing samples of different concentrations. The positive control group was cultured with primary differentiation medium containing positive drugs, and the blank control group was cultured with normal complete medium. Then, the culture medium of the model group was replaced with secondary differentiation medium, and the test group and the positive control group were cultured with secondary differentiation medium containing samples of different concentrations and positive drugs, and the blank control group was still cultured with normal complete medium. The secondary differentiation medium was a complete medium containing 10μg / mL insulin. Each group of cells was cultured continuously for 4 days, and the secondary differentiation medium was updated once a day. After that, all of them were replaced with normal complete medium until 80% of the cells differentiated into mature adipocytes, which was the completion of differentiation.

[0083] 2. Oil Red O Staining

[0084] After differentiation is complete, discard the old culture medium, wash the cells with PBS buffer, and fix the cells with 4% paraformaldehyde solution for 10 minutes at room temperature, and wash twice with PBS. Stain according to the instructions of the Oil Red O staining kit, add staining solution and cover the cells for 20 seconds. After removing the wash solution, add an appropriate amount of Oil Red O staining solution, stain for 30 minutes, remove the staining solution, wash with staining solution for 30 seconds, wash with PBS for 20 seconds, and discard PBS. Cover the cells evenly with PBS again, observe and take pictures under a microscope, and the results are as follows Figure 7 After taking the photo, discard the PBS, add 500 μL of isopropanol to the 24-well plate and shake for 5 minutes, and measure the absorbance at a wavelength of 520 nm. The results are shown in Figure 8 shown.

[0085] from Figure 7It can be seen that compared with the model group, the number of lipid droplets in the differentiated cells added with the buckwheat protein-derived lipid-lowering peptide of the present invention is reduced, and as the concentration increases, the lipid droplets in the cells gradually decrease. Figure 8 It can be seen that FA-8, FE-10 and LA-9 can significantly reduce lipid accumulation in cells, which are reduced by 39.30±9.74%, 19.19%±6.99% and 29.52±10.02% respectively compared with the model group. The above shows that the bitter buckwheat protein-derived lipid-lowering peptides of the present invention can inhibit the differentiation of preadipocytes.

[0086] 3.3 Determination of TC, TG, HDL-C and LDL-C contents

[0087] After differentiation, the old culture medium was discarded, the cells were washed once with PBS, and the cells were collected according to the instructions of the TG kit (A110-1-1, Nanjing Jiancheng Bioengineering Institute), TC kit (A111-1-1, Nanjing Jiancheng Bioengineering Institute), HDL-C kit (A112-1-1, Nanjing Jiancheng Bioengineering Institute) and LDL-C kit (A113-1-1, Nanjing Jiancheng Bioengineering Institute), and the cells were broken with lysis buffer. Finally, the TG, TC, HDL-C and LDL-C contents in the cells were measured. The results are shown in Fig. 9 shown.

[0088] from Fig. 9 A It can be seen that at a concentration of 40 μg / mL, the three buckwheat protein-derived lipid-lowering peptides FA-8, FE-10 and LA-9 can significantly reduce the content of TG in cells, which was decreased by 35.38%±17.39%, 43.95%±2.28% and 53.75%±2.25% respectively compared with the model group.

[0089] from Fig. 9 B It can be seen that at a concentration of 40 μg / mL, the three buckwheat protein-derived lipid-lowering peptides FA-8, FE-10 and LA-9 can significantly reduce the content of TC in cells, which was decreased by 51.46%±3.07%, 56.01%±7.38% and 68.58±4.17% respectively compared with the model group.

[0090] from Fig. 9 C It can be seen that at a concentration of 40 μg / mL, the three buckwheat protein-derived lipid-lowering peptides FA-8, FE-10 and LA-9 can significantly increase the content of HDL-C in cells, which is increased by 108.80%±25.40%, 219.20%±58.39% and 289.60%±39.97% respectively compared with the model group.

[0091] from Fig. 9D It can be seen that at a concentration of 40 μg / mL, the three buckwheat protein-derived lipid-lowering peptides, FA-8, FE-10 and LA-9, can significantly reduce the content of LDL-C in cells, which was decreased by 55.00%±18.16%, 70.50%±10.89% and 66.00%±8.84% respectively compared with the model group.

[0092] The above results show that the bitter buckwheat protein-derived lipid-lowering peptide of the present invention has the ability to lower total cholesterol, lower triglycerides, lower high-density lipoprotein cholesterol and increase high-density lipoprotein cholesterol.

[0093] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A lipid-lowering peptide derived from tartary buckwheat protein, characterized in that: The tartary buckwheat protein-derived lipid-lowering peptide is at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

2. Use of the tartary buckwheat protein-derived lipid-lowering peptide according to claim 1 in the preparation of a medicament for preventing and / or treating diseases related to hyperlipidemia.

3. The use according to claim 2, characterized in that: The hyperlipidemia-related disease is obesity and / or hyperlipidemia.

4. The use according to claim 2, characterized in that: The drug is a drug for inhibiting the differentiation of preadipocytes.

5. The use according to claim 2, characterized in that: The drug is a drug for lowering total cholesterol.

6. The use according to claim 2, characterized in that: The drug is a drug for lowering triglycerides.

7. The use according to claim 2, characterized in that: The drug is a drug for lowering high-density lipoprotein cholesterol.

8. The use according to claim 2, characterized in that: The drug is a drug for increasing high-density lipoprotein cholesterol.

9. The use according to claim 2, characterized in that: The drug is a drug that inhibits the activity of pancreatic lipase.

10. Use of the tartary buckwheat protein-derived lipid-lowering peptide according to claim 1 in the preparation of a health product for assisting in lowering blood lipids.

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