A tartary buckwheat protein-derived lipid-lowering peptide and its application

Abstract

The present invention relates to a tartary buckwheat protein-derived lipid-lowering peptide and its use. The tartary buckwheat protein-derived lipid-lowering peptide has one or more of the following sequences: FHWDYPQA; FHWDYPQALE; LFHWDYPQA. This tartary buckwheat protein-derived lipid-lowering peptide has excellent pancreatic lipase inhibition, effectively inhibiting preadipocyte differentiation, and has the ability to lower total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while increasing high-density lipoprotein cholesterol. Furthermore, the tartary buckwheat protein-derived lipid-lowering peptide is non-toxic and highly safe.

Description

Technical Field

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

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

[0003] Bioactive peptides are protein fragments that positively impact bodily functions, regulating and influencing health. Many bioactive peptides from animal and plant sources have demonstrated promising lipid-lowering activity. Tartary buckwheat is widely used for its nutritional value and beneficial health effects, particularly in the treatment of chronic conditions such as diabetes, obesity, and cardiovascular disease. For example, Ma et al. (DOI: 10.1016 / j.foodchem.2005.01.052) isolated a peptide from common buckwheat that inhibits angiotensin I-converting enzyme activity. However, research on active buckwheat protein peptides remains limited.

[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 objective of the present invention is to overcome the aforementioned lack of existing active buckwheat protein peptides with lipid-lowering activity by providing a buckwheat protein-derived lipid-lowering peptide. This buckwheat protein-derived lipid-lowering peptide exhibits excellent pancreatic lipase inhibition, effectively inhibits preadipocyte differentiation, and has the ability to lower total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while increasing high-density lipoprotein cholesterol. Furthermore, the buckwheat protein-derived lipid-lowering peptide 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 medicament 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 health products that assist in lowering blood lipids.

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

[0009] The above-mentioned purpose of the present invention is achieved through 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. 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] Through research, the inventors of the present invention have discovered that the tartary buckwheat protein-derived lipid-lowering peptide of the present invention has excellent pancreatic lipase inhibition, can effectively inhibit the differentiation of preadipocytes, and has the ability to lower total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while increasing high-density lipoprotein cholesterol. Furthermore, the tartary buckwheat protein-derived lipid-lowering peptide of the present invention is non-toxic and highly safe.

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

[0014] Preferably, the tartary buckwheat protein-derived lipid-lowering peptide is the 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 the 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 lowering 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 peptides in the preparation of drugs for preventing and / or treating diseases related to hyperlipidemia is also within the scope of protection 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 that increases 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 an injection, a tablet, an oral solution, a granule or a capsule.

[0026] The present invention also protects the use of the tartary buckwheat protein-derived lipid-lowering peptide in the preparation of health-care products 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 buckwheat protein-derived lipid-lowering peptide of the present invention has excellent pancreatic lipase inhibition, can effectively inhibit the differentiation of preadipocytes, and has the ability to lower total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while increasing high-density lipoprotein cholesterol. Furthermore, the 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 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; Figure 2 A is the elution peak 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 buckwheat protein-derived lipid-lowering peptides and pancreatic lipase (PDB: 1LPB).

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

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

[0036] Figure 6 The figure shows the toxicity test results of three buckwheat protein-derived lipid-lowering 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 520 nm of three buckwheat protein-derived lipid-lowering peptides and orlistat after lipid droplets stained with Oil Red O were dissolved in isopropanol.

[0039] Figure 9 This is a graph showing the lipid-lowering activity assay of buckwheat protein-derived lipid-lowering peptides in 3T3-L1 preadipocytes; Figure 9 A is the effect diagram on TG content; Figure 9 B is the effect diagram on TC content; Figure 9 C is the effect diagram on HDL-C content; Figure 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 determination method of pancreatic lipase (PL) inhibition rate of the present invention is as follows:

[0042] A 1 mg / mL solution of 4-nitrophenyl laurate was used as the reaction substrate, which contained 0.05 mol / 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 later use. The reaction substrate, the sample solution to be tested, and the reaction buffer (pH 8.2, 0.1 mol / L Tris-HCl buffer) were added to a 2 mL test tube in sequence. Finally, a pancreatic lipase solution (5 mg / mL) was added to initiate the reaction. The volume ratio of the added samples was 5:2:4:3. After incubation at 37°C for 2 hours, the absorbance was measured at a wavelength of 420 nm. A sample group without enzyme and a blank group without sample were also 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 methods of various embodiments of the present invention are as follows:

[0045] The data were plotted using Prism 8.0.2 and Origin 2019, and statistically analyzed using SPSS 21.0 and Excel 2021. The results are expressed as mean ± standard deviation (SD). The ANOVA method was used to analyze the significance of differences between samples (P < 0.05). Each experiment was repeated three times.

[0046] Example 1 Extraction and enzymatic hydrolysis of tartary buckwheat protein, and separation and purification of the enzymatic hydrolysate

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

[0048] Buckwheat (Xiqiao No. 8, provided by Xichang University) was ground, passed through a 100-mesh sieve, and defatted for 2 h with petroleum ether at a solid-liquid ratio of 1:5 (g / mL). Degreasing was repeated twice and the mixture was air-dried in a fume hood to obtain defatted buckwheat powder. Based on the principle of alkali dissolution and acid precipitation, the defatted buckwheat powder was mixed with water at a ratio of 1:10 (g / mL). The pH was adjusted to 9.0 with 0.1 mol / L NaOH, and the mixture was stirred in a 45°C water bath for 2 h. The mixture was centrifuged at 4000 rpm for 20 min. The supernatant was collected, and the pH was adjusted to 4.4 with 0.1 mol / L HCl. The supernatant was allowed to stand at 4°C for 2 h to precipitate the protein. The protein was then centrifuged at 4000 rpm for 20 min. The resulting precipitate was the crude buckwheat protein, and freeze-dried for later use.

[0049] A 3% crude buckwheat protein solution was prepared, and the optimal enzymatic hydrolysis process of the document "Enzymatic Preparation, Amino Acid Composition and Activity of Buckwheat Protein-derived Lipid-lowering Peptides" (DOI: 10.13386 / j.issn1002-0306.2023110029) was referred to. Bromelain was used for enzymatic hydrolysis, and the enzyme was inactivated at 95°C. After cooling, the solution was centrifuged at 8000 rpm for 20 min. 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 ultrafiltration 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, and 4 mg / mL). The pancreatic lipase inhibition rate and IC50 value were determined. The results are shown in Figure 2. Figure 1 shown.

[0052] The pancreatic lipase inhibition ability of the two ultrafiltration fractions MW < 3 kDa, MW > 3 kDa and the hydrolysate is as follows Figure 1 As shown. At each mass concentration, the pancreatic lipase inhibition rate of the MW < 3kDa fraction was significantly higher than that of the hydrolysate fraction and the MW > 3kDa fraction. At a concentration of 4 mg / mL, the inhibition rate of the MW < 3kDa fraction 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 concentration was only 0.019 mg / mL. Therefore, the fraction with MW < 3 kDa was selected for further purification.

[0053] The ultrafiltration fraction with a MW <3 kDa was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The ultrafiltration fraction was filtered through a 0.22 μm membrane and separated using a C18 reversed-phase column (20 mm × 450 mm, 10 μm). Nine fractions were isolated and designated P1 to P9. 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%-50% mobile phase B; 40-80 min, 50%-75% mobile phase B; 80-85 min, 75%-90% mobile phase B; 85-95 min, 90%-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 concentration to determine their pancreatic lipase inhibitory ability and screen out the component 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 < 3kDa fraction separated by RP-HPLC and the pancreatic lipase inhibitory ability of the P1 to P9 fractions. Figure 2 As can be seen, fraction P9 has the highest pancreatic lipase inhibitory activity. At a concentration of 1 mg / mL, its pancreatic lipase inhibition rate reached 68.23% ± 0.98%, significantly higher than that of other fractions. Therefore, we selected fraction P9 for subsequent peptide composition identification.

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

[0056] 1. Identification of P9 components by high performance liquid chromatography-tandem mass spectrometry

[0057] The P9 fraction was analyzed by LC-MS / MS equipped with an online nanospray ionization source. The system consisted of an Orbitrap Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, MA, USA) connected to an EASY-nanoLC1200. A total of 1 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm x 25 cm. The sample was separated using a 60-min gradient with a controlled column flow rate of 300 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started with 2% phase B and was increased nonlinearly to 35% at 47 min, then to 100% over 1 min, where it was 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 using PEAKSStudio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKSDB was used to search the uniprot-Fagopyrum tataricum database (version 2023, 382 entries) with a "none" digestion setting. Search parameters included a fragment ion mass tolerance of 0.02 Da, a precursor mass tolerance of 10 ppm, variable modifications: oxidation (M) 15.99, destruction (NQ) 0.98, and a protein cardiology score of at least one unique peptide; a peptide cardiology score of -101 gP ≥ 20.

[0060] A total of 2,662 peptide sequences were identified for the P9 component using the above method, of which 40 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. The original ligand methoxyundecylphosphonic acid (MUP) and metal ion of the receptor protein 1LPB were separated and docked. The receptor protein 1F6W does not contain the original ligand and can be docked directly. Next, the protein receptor was dehydrated and hydrogenated, and 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 for the peptide to 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, and number modes = 10. The docking parameters for the peptide to 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, and number modes = 10. The docked conformation with the lowest binding energy was selected as the optimal conformation. A three-dimensional structure diagram of the peptide-enzyme interaction was created using PYMOL and visualized using Discovery Studio 2019. A two-dimensional interaction map was generated to analyze the interaction types after docking.

[0063] The 40 peptides were ranked according to their molecular docking binding energies, with lower binding energies indicating better binding. Three tartary buckwheat protein-derived lipid-lowering peptides were identified: FHWDYPQA, FHWDYPQALE, and LFHWDYPQA. These peptides will be subsequently named using the initial and final letters of their sequences and lengths, namely FA-8, FE-10, and LA-9. The identification results and peptide characteristics of these tartary buckwheat protein-derived lipid-lowering peptides 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 3As can be seen, FA-8 forms a salt bridge with 1LPB at amino acid residue ASP328, an electrostatic interaction with amino acid residue ASP12, and π-anionic electrostatic interactions with amino acid residues ARG337 and LYS367. It also forms hydrogen bonds with amino acid residues SER47, ARG44, ASP389, ARG337, ASP387, ASN229, ASP328, LYS367, ASP331, and GLU13. It also forms a carbon-hydrogen bond with amino acid residue ALA43. It also forms an amide-π stacking hydrophobic interaction with amino acid residue GLY236, and π-alkyl interactions with amino acid residues ALA332, ALA40, LYS239, and PRO235.

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

[0068] LA-9 forms salt bridges with 1LPB at amino acid residues ASP387 and ASP331, generates electrostatic interactions with amino acid residue ASP12, forms π-anionic interactions with amino acid residue LYS24, forms hydrogen bonds with amino acid residues GLU64, THR292, ASP331, ASP387, ASN294, ASP328, ARG339, ARG337, LYS367, ALA332, and ASN384, forms π-sigma hydrophobic interactions with amino acid residue LEU41, forms amide-π stacking hydrophobic interactions with amino acid residue GLU13, and forms 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 forms hydrogen bonds with 1F6W at amino acid residues ASN232, VAL285, PHE351, LYS231, and PRO226, forms a carbon-hydrogen bond with amino acid residue LYS231, forms π-cationic electrostatic interactions with amino acid residues LYS355 and LYS231, forms π-sigma hydrophobic interactions with amino acid residues HIS283 and ILE301, and forms 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 show that the three buckwheat protein-derived lipid-lowering peptides bind to the receptor through hydrogen bonds, π-π bonds, hydrophobic, electrostatic and other interactions. These interactions will enhance the binding of peptides and receptor proteins or change the spatial conformation of the enzyme, thereby affecting its normal physiological function 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, responsible for digesting nearly 70% of dietary fat intake. Three tartary buckwheat protein-derived lipid-lowering peptides, FA-8, FE-10, and LA-9, obtained through molecular docking, were prepared into test sample solutions at different concentrations (50, 100, 200, 400, and 800 μg / mL). Orlistat, a key drug for treating obesity, was used as a positive control to determine the inhibition rate of pancreatic lipase. The results are shown in Figure 2. Figure 5 shown.

[0075] from Figure 5 The results show that the buckwheat protein-derived lipid-lowering peptides exhibited strong inhibitory effects on pancreatic lipase at concentrations ranging from 50 to 800 μg / mL, with a concentration-dependent inhibition. LA-9 (LFHWDYPQA) exhibited the highest inhibition rate at 800 μg / mL, reaching 83.17% ± 0.52%. FA-8 and FE-10 were second, with inhibition rates of 78.52% ± 0.29% and 75.59% ± 0.55%, respectively. The inhibitory rates of the three buckwheat protein-derived lipid-lowering peptides on pancreatic lipase were higher than those of existing bioactive peptides, such as adzuki bean protein hydrolysate IFNNDPNNHP, which exhibited a 62.60% inhibition rate 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 monomers was 98%, and their 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 continuing to culture for 24 hours, 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 6The figure shows the toxicity test results of buckwheat protein-derived hypolipidemic peptide and orlistat. Figure 6 It can be seen that when the concentrations of buckwheat protein-derived lipid-lowering peptide and orlistat are 1-80 μg / mL, the cell survival rates are all 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. High-fat model differentiation induction

[0082] 3T3-L1 preadipocytes were seeded in 24-well plates (5 × 10 4 Cells were 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 due to 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 at 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. Next, 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 at different concentrations and positive drugs, while the blank control group was still cultured with normal complete medium. The secondary differentiation medium was a complete medium containing 10μg / mL insulin. The cells in each group were cultured continuously for 4 days, and the secondary differentiation medium was renewed once a day. Afterwards, all the cells were replaced with normal complete medium until 80% of the cells differentiated into mature adipocytes, which was considered to be 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, then wash with PBS for 20 seconds, and discard PBS. Cover the cells evenly with PBS again, observe under a microscope and take pictures. 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 the figure. Figure 8 shown.

[0085] from Figure 7It can be seen that compared with the model group, the number of lipid droplets in the cells differentiated by adding 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 As can be seen, FA-8, FE-10, and LA-9 can all significantly reduce lipid accumulation in cells, with reductions of 39.30±9.74%, 19.19%±6.99%, and 29.52±10.02%, respectively, compared to the model group. This indicates that the 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 and the cells were washed once with PBS. 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 disrupted with lysis buffer. Finally, the TG, TC, HDL-C, and LDL-C levels in the cells were measured. The results are shown in Figure 2. Figure 9 shown.

[0088] from Figure 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 TG content in cells, which decreased by 35.38%±17.39%, 43.95%±2.28% and 53.75%±2.25% respectively compared with the model group.

[0089] from Figure 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 decreased by 51.46%±3.07%, 56.01%±7.38% and 68.58±4.17% respectively compared with the model group.

[0090] from Figure 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 are increased by 108.80%±25.40%, 219.20%±58.39% and 289.60%±39.97% respectively compared with the model group.

[0091] from Figure 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 LDL-C content in cells, which 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 tartary 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 the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection 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, characterized in that: The hyperlipidemia-related disease is obesity and / or hyperlipidemia.

3. The use according to claim 2, characterized in that The drug is a drug that inhibits the differentiation of preadipocytes.

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

5. The use according to claim 2, characterized in that The drug is a triglyceride-lowering drug.

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

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

8. The use according to claim 2, characterized in that The drug is a drug that inhibits pancreatic lipase activity.

9. 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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