A hypolipidemic peptide and its application

Abstract

The present invention relates to a lipid-lowering peptide and its application. The sequence of the lipid-lowering peptide is: QIF (Gln-Ile-Phe). This lipid-lowering peptide not only has strong bile salt-binding ability, pancreatic lipase inhibitory effect and cholesterol esterase inhibitory effect, but also can effectively reduce the lipid content in cells, reduce the low-density lipoprotein content in cells and increase the high-density lipoprotein content in cells, thus having excellent lipid-lowering activity and can be widely used in lipid-lowering drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and more particularly, to a lipid-lowering peptide and its application. Background Art

[0002] With the economic development and the improvement of people's living standards, some unhealthy living habits have emerged, such as sedentary lifestyle, increased high-energy diet, and reduced physical activity. These unhealthy habits are likely to lead to metabolic disorders and functional impairments in the body, and then trigger some metabolic syndromes, such as the prevalent obesity, hypertension, hyperglycemia, hyperlipidemia, etc. Hyperlipidemia is a disease caused by abnormal levels of lipids or lipoproteins in the blood and is a major risk factor for inducing metabolic syndrome and known cardiovascular diseases. Hyperlipidemia is mainly manifested by abnormal levels of plasma total cholesterol, total triglycerides, low-density lipoprotein, and high-density lipoprotein.

[0003] At present, there are some chemical drugs on the market for the treatment of hyperlipidemia, such as statins and fibrates. Although these drugs can effectively regulate lipid abnormalities and reduce plasma cholesterol, they are expensive and usually have varying degrees of toxic side effects. Therefore, the development of safe and effective natural lipid-lowering components has become the focus of current attention.

[0004] Tea contains a large number of substances beneficial to human health. Long-term drinking helps to delay aging and reduce blood pressure and lipids. The Chinese patent named Preparation of a Rosa roxburghii Tratt, edible mushroom and tea fermented beverage for lowering blood lipid and blood sugar reduces blood lipid through the compound fermentation of Rosa roxburghii Tratt, edible mushroom and tea, but it does not study which bioactive components in the compound fermentation of tea play a role in reducing blood lipid.

[0005] Therefore, it is necessary to research and find lipid-lowering bioactive components derived from tea. Summary of the Invention

[0006] The primary object of the present invention is to overcome the problem of insufficient research on the lipid-lowering bioactive components of tea in the above-mentioned prior art and provide a lipid-lowering peptide. This lipid-lowering peptide not only has strong bile salt-binding ability, pancreatic lipase inhibitory effect, and cholesterol esterase inhibitory effect, but also can effectively reduce the lipid content in cells, reduce the low-density lipoprotein content in cells, and increase the high-density lipoprotein content in cells, thus having excellent lipid-lowering activity and can be widely applied to lipid-lowering drugs.

[0007] A further object of the present invention is to provide the application of the above lipid-lowering peptide in the preparation of lipid-lowering drugs.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] A lipid-lowering peptide, the sequence of the lipid-lowering peptide is: QIF.

[0010] The inventors of the present invention extracted a lipid-lowering peptide from tea residues, and the sequence of the lipid-lowering peptide is: QIF (Gln-Ile-Phe). Through research by the inventors of the present invention, it was found that this lipid-lowering peptide not only has strong bile salt binding ability, pancreatic lipase inhibitory effect and cholesterol esterase inhibitory effect, but also can effectively reduce the lipid content in cells, reduce the low-density lipoprotein content in cells and increase the high-density lipoprotein content in cells, thus having excellent lipid-lowering activity and can be widely used in lipid-lowering drugs.

[0011] The application of the above lipid-lowering peptide in the preparation of lipid-lowering drugs is also within the protection scope of the present invention.

[0012] Preferably, it is the application of the lipid-lowering peptide in the preparation of drugs for reducing the lipid content in cells.

[0013] Preferably, it is the application of the lipid-lowering peptide in the preparation of drugs for reducing the low-density lipoprotein content in cells and increasing the high-density lipoprotein content in cells.

[0014] Preferably, it is the application of the lipid-lowering peptide in the preparation of drugs for promoting cholesterol degradation and metabolism.

[0015] More preferably, it is the application of the lipid-lowering peptide in the preparation of drugs that bind to bile salts.

[0016] Preferably, it is the application of the lipid-lowering peptide in the preparation of drugs for inhibiting the activity of pancreatic lipase.

[0017] More preferably, it is the application of the lipid-lowering peptide in the preparation of drugs that bind to the amino acid residues Ser152, Phe77, His151, Tyr114 and Gly76 of pancreatic lipase through hydrogen bonds.

[0018] More preferably, it is the application of the lipid-lowering peptide in the preparation of drugs that bind to the amino acid residue Arg256 of pancreatic lipase through electrostatic interaction.

[0019] More preferably, it is the application of the lipid-lowering peptide in the preparation of drugs that bind to the amino acid residues Ile78, Ala259, Phe215 and Ala260 of pancreatic lipase through hydrophobic interaction.

[0020] Preferably, it is the application of the lipid-lowering peptide in the preparation of drugs for inhibiting the activity of cholesterol esterase.

[0021] More preferably, it is the application of the lipid-lowering peptide in the preparation of drugs that bind to the amino acid residue Gly106 of cholesterol esterase through hydrogen bonds.

[0022] More preferably, the application of the lipid-lowering peptide in the preparation of a drug that binds to the amino acid residues Tyr105, Tyr125, Ile439, Val285, Leu282, Trp227, Ser194, Phe393, Ala195, His435, Phe324, Ala436, Ile323, Gly107, and Glu193 of cholesterol esterase through van der Waals forces.

[0023] More preferably, the application of the lipid-lowering peptide in the preparation of a drug that binds to the amino acid residues Met281, Leu392, Met111, Ala108, and Leu110 of cholesterol esterase through hydrophobic interactions.

[0024] Preferably, the application of the lipid-lowering peptide in the preparation of a drug for inhibiting adipogenic differentiation of cells.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The lipid-lowering peptide of the present invention not only has strong bile salt-binding ability, pancreatic lipase inhibitory effect, and cholesterol esterase inhibitory effect, but also can effectively reduce the lipid content in cells, reduce the low-density lipoprotein content in cells, and increase the high-density lipoprotein content in cells. Therefore, it has excellent lipid-lowering activity and can be widely used in lipid-lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph showing the bile salt-binding ability of the hydrolysates obtained by enzymatically hydrolyzing the crude tea residue protein in Example 1 with different tool enzymes and hydrolysis times. Among them, Figure 1 A is a graph showing the bile salt-binding ability of the hydrolysates obtained by enzymatically hydrolyzing with four tool enzymes against 3 bile salts; Figure 1 B is a graph showing the bile salt-binding ability of the hydrolysates obtained with pepsin as the tool enzyme at different hydrolysis times.

[0028] Figure 2 A is a graph showing the bile salt-binding ability of the tea protease hydrolysate in Example 1 and different ultrafiltration fractions; Figure 2 B is a chromatogram of the <3 kDa fraction in Example 1 separated and purified by preparative liquid chromatography; Figure 2 C is the test result of the pancreatic lipase inhibitory activity and cholesterol esterase inhibitory activity of the F6 fraction.

[0029] Figure 3 It is a molecular docking result graph of the lipid-lowering peptide and pancreatic lipase. Among them, Figure 3 A is a 3D graph of the molecular docking result of the lipid-lowering peptide and pancreatic lipase, Figure 3 B is a 2D graph of the molecular docking result of the lipid-lowering peptide and pancreatic lipase.

[0030] Figure 4 It is a molecular docking result diagram of lipid-lowering peptide and cholesterol esterase. Among them, Figure 4 A is a 3D diagram of the molecular docking result of lipid-lowering peptide and cholesterol esterase, Figure 4 B is a 2D diagram of the molecular docking result of lipid-lowering peptide and cholesterol esterase.

[0031] Figure 5 It is a result diagram of the in vitro lipid-lowering activity of lipid-lowering peptide.

[0032] Figure 6 It is a test result diagram of the effect of lipid-lowering peptide on lipid accumulation in 3T3-L1 preadipocytes.

[0033] Figure 7 It is a test result diagram of the effect of lipid-lowering peptide on lipoprotein levels in adipocytes. Detailed implementation manners

[0034] In order to describe the technical solutions of the present invention more clearly and completely, the present invention will be further described in detail below through specific embodiments. 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, and various changes can be made within the scope defined by the claims of the present invention.

[0035] The main materials and reagents used in each embodiment are described as follows:

[0036] Tea residues are provided by the Key Laboratory of Tea Science in Yunnan Province;

[0037] Pepsin (3×10 6 U / mg), neutral protease (1×10 5 U / g), trypsin (2.5×10 5 U / g) and papain (2×10 5 U / g) are purchased from Nanning Pangbo Bioengineering Co., Ltd.;

[0038] Sodium taurocholate (STC), porcine pancreatic lipase, p-nitrophenyl laurate (pNP laurate), p-nitrophenyl butyrate (PNPB), orlistat, simvastatin, thiazolyl blue (MTT), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX) and insulin are purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0039] Cholesterol esterase and cholestyramine are purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0040] 3T3-L1 preadipocytes are from the Cell Bank of the Chinese Academy of Sciences;

[0041] DMEM (dulbecco's modified eagle medium) medium, fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco, USA;

[0042] Oil Red O kit, high-density lipoprotein (HDL-c) kit, and low-density lipoprotein (LDL-c) kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0043] All other chemicals and reagents were of analytical grade.

[0044] Example 1 Obtaining Hypolipidemic Peptides

[0045] 1.1 Preparation of Tea Protease Hydrolysate

[0046] Bile acids are the main components of bile, the products of cholesterol metabolism and decomposition in the liver, and also the main way for the human body to remove cholesterol. Most bile acids in the body exist in the form of bile salts. Therefore, substances with a certain bile salt-binding ability can be identified as having certain hypolipidemic activity.

[0047] The process for evaluating bile salt-binding ability is as follows: Add 1 mL of 0.01 mol / L HCl solution to 1 mL of the sample and incubate at 37 °C for 1 h; after adjusting the pH to 6.3 with 0.1 mol / L NaOH, add 5 mL of bile salt standard solutions with a concentration of 1 mmol / L (bile salt standard solutions include sodium glycocholate (SGC) standard solution, sodium cholate (SC) standard solution, and sodium taurocholate (STC) standard solution). In the blank group, replace the sample with phosphate buffer. After reacting at 37 °C for 1 h, centrifuge at 4000 r / min for 20 min, take 2.5 mL of the supernatant, add 7.5 mL of 60% sulfuric acid solution, incubate at 70 °C for 30 min, then cool to room temperature, and finally measure the absorbance at a wavelength of 387 nm. Calculate the bile salt-binding ability through the absorbance value and the bile salt standard curve, and calculate according to the following formula:

[0048]

[0049] where C 0 is the concentration of bile salts in the supernatant of the blank group, and C 1 is the concentration of bile salts in the supernatant after adding the sample.

[0050] Take a certain amount of tea residues and add them to 0.1 mol / L NaOH solution at a liquid-solid ratio of 1:40 (g:mL), and then extract at a constant temperature of 40 °C in a water bath for 4 h. After the extraction, filter and centrifuge, and collect the supernatant; adjust the pH of the supernatant to 4.5 with 1 mol / L hydrochloric acid, let it stand for 20 min, then centrifuge at a speed of 4000 r / min for 20 min, collect the precipitate, and freeze-dry to obtain the crude tea residue protein extract.

[0051] Select trypsin (pH 8.0, temperature 37 °C), papain (pH 6.5, temperature 55 °C), pepsin (pH 1.5, temperature 37 °C) and neutral protease (pH 7.0, temperature 45 °C) as tool enzymes, and add them to the aqueous solution of the crude tea residue protein extract with a concentration of 3 g / mL at an enzyme-substrate ratio of 0.3%. After enzymatic hydrolysis for 3 h, an enzymatic hydrolysate is obtained. Taking the bile salt binding ability as an index, the best tool enzyme is determined. The bile salt binding abilities of the enzymatic hydrolysates obtained by enzymatic hydrolysis with the four proteases are as Figure 1 shown in Figure 1 Figure A. As can be seen from

[0052] Figure A, the enzymatic hydrolysate obtained by pepsin shows the highest bile salt binding ability, and its binding rates to sodium cholate (SC), sodium taurocholate (STC) and sodium glycocholate (SGC) are 63.84%, 65.91% and 59.14% respectively. Figure 1 shown in Figure 1 Figure B. As can be seen from

[0053] 1.2 Isolation, purification and identification of hypolipidemic peptides

[0054] Ultrafiltration separation of the tea protease hydrolysate is carried out using an ultrafiltration membrane with a molecular weight cut-off of 3 kDa to obtain two ultrafiltration components: ≥3 kDa component and <3 kDa component. Freeze-dry the ultrafiltration components and measure their bile salt binding abilities respectively. The bile salt binding abilities of the tea protease hydrolysate and the two ultrafiltration components are as Figure 2 shown in Figure 2 Figure A. As can be seen from

[0055] Figure A, at a concentration of 1 mg / mL, the bile salt binding activity of the <3 kDa component has a significant improvement compared with the tea protease hydrolysate, and its abilities to bind STC and SGC are as high as 36.61% and 38.76% respectively. Therefore, the <3 kDa component is selected for further purification in the follow-up.Reverse high performance liquid chromatography separation and purification: After the fraction less than 3 kDa obtained by ultrafiltration was passed through a 0.45 μm filter membrane, preparative liquid chromatography separation was carried out using a C18 reverse phase chromatographic column (20 mm × 450 mm, 10 μm). 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, 6% - 25% mobile phase B; 40 - 70 min, 25% - 70% mobile phase B; 70 - 75 min, 70% - 90% mobile phase B; 75 - 85 min, 90% - 90% mobile phase B; injection volume 5 mL; flow rate 10 mL / min; detection wavelengths were 214 nm and 280 nm. The fractions corresponding to each elution peak were collected, evaporated to concentrate and freeze-dried, and their bile salt binding abilities were detected respectively to screen the fraction with the strongest lipid-lowering activity. The chromatogram of the fraction less than 3 kDa separated and purified by preparative liquid chromatography is shown in Figure 2 Figure B. A total of 7 fractions were separated, named F1 to F7 respectively, and their bile salt binding abilities were detected respectively. Among them, F6 had the strongest bile salt binding ability. At a concentration of 1 mg / ml, its binding rates to STC and SGC were 48.73% and 49.79% respectively. Subsequently, the in vitro lipid-lowering activities of the tea protease hydrolysate and fraction F6 were compared and analyzed from the perspectives of pancreatic lipase inhibitory activity and cholesterol esterase inhibitory activity.

[0056] The process for determining the pancreatic lipase inhibitory activity was as follows: Porcine pancreatic lipase was prepared into 5 mg / ml with primary water. A certain amount of the reaction substrate p-nitrophenyl laurate (pNP laurate) was dissolved in an aqueous sodium acetate solution containing 1% Triton X-100 to prepare a 0.1% (w / v) pNP laurate solution. 250 μL of the reaction substrate, 100 μL of the sample solution and 200 μL of the reaction buffer were added to a centrifuge tube, and finally 150 μL of the pancreatic lipase solution was added to initiate the reaction. After reacting at 37 °C for 2 h, it was centrifuged at 10000 rpm for 1 min, and the absorbance value of the supernatant was detected at a wavelength of 420 nm. For the blank control, the sample solution was replaced with the buffer solution, and for the sample control, the pancreatic lipase solution was replaced with the reaction buffer solution. The pancreatic lipase inhibition rate was calculated according to the following formula:

[0057]

[0058] In the formula: A 1 is the absorbance value of the sample supernatant; A 2 is the absorbance value of the sample control supernatant; A 0 is the absorbance value of the blank control supernatant.

[0059] The process for determining the cholesterol esterase inhibitory activity is as follows: Porcine cholesterol esterase is prepared with primary water at a concentration of 4.2 μg / mL. All reactions are carried out in a sodium phosphate buffer (0.1 mol / L, pH 7.0) containing NaCl (0.1 mol / L), p-nitrophenyl butyrate (PNPB, 0.2 mmol / L), and sodium taurocholate (STC, 5.16 mmol / L). Add 10 μL of the reaction substrate PNPB, 25 μL of the sample solution, and 1 mL of the buffer into a centrifuge tube. Finally, add 50 μL of the cholesterol esterase solution to initiate the reaction. React at 25.0 °C for 5 min and measure the absorbance at 405 nm. The blank tube uses primary water instead of the sample solution, the blank control tube uses primary water instead of the cholesterol esterase solution and the sample solution, and the background control tube uses primary water instead of the cholesterol esterase solution. Calculate the cholesterol esterase inhibitory activity according to the following formula:

[0060]

[0061] In the formula: A is the absorbance of the blank tube; B is the absorbance of the blank control tube; C is the absorbance of the sample tube; D is the absorbance of the background control tube.

[0062] The test results of pancreatic lipase inhibitory activity and cholesterol esterase inhibitory activity are as Figure 2 shown in C. As can be seen from Figure 2 C, the IC 50 values of the inhibitory effects of the F6 fraction on pancreatic lipase and cholesterol esterase are 0.153 mg / mL and 0.549 mg / mL, respectively, which are only about half of the IC 50 of the protease hydrolysis of tea residue. This result further indicates that after liquid chromatography separation, the high-activity hypolipidemic components have been effectively enriched and separated. Therefore, a large amount of the F6 fraction is collected for the subsequent identification of polypeptide composition and amino acid sequence.

[0063] The F6 fraction obtained by ultrafiltration and reverse high performance liquid chromatography separation and purification of tea protease hydrolysate was identified by HPLC-MS / MS. The process of HPLC-MS / MS identification was as follows: The F6 fraction was used as a sample, and the sample was analyzed by LC-MS / MS equipped with an online nanoelectrospray ionization source. The whole system was a QExactive mass spectrometer (Thermo Fisher Scientific, MA, USA) in series with an EASY-nanoLC1200. A total of 10 μL of the sample was loaded (analysis column: Acclaim PepMap C18, 75 μm x 25 cm), the column flow rate was 300 nL / min, the column temperature was 40 °C, and the electrospray voltage was 2 kV. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was ACN solution containing 0.1% formic acid. The chromatographic gradient was as follows: 0 - 3 min, 2% - 6% mobile phase B; 3 - 42 min, 6% - 20% mobile phase B; 42 - 47 min, 20% - 35% mobile phase B; 47 - 48 min, 35% - 100% mobile phase B; 48 - 100 min, 100% - 100% mobile phase B. The mass spectrometry parameters were set as follows: (1) MS: The scanning range (m / z) was 200 - 1500; the resolution was 70,000; the AGC target was 3e6; the maximum injection time was 60 ms; the scanning charge was 1 - 7; (2) HCD-MS / MS (top10): The resolution was 17,500; the isolation window was 2.2 m / z; the AGC target was 5e4; the maximum injection time was 50 ms; the collision energy was 27, and the dynamic exclusion time was 20 s. After identification by HPLC-MS / MS, a total of 125 peptide sequences were identified. Among them, there were 37 small peptides with a peptide segment length not exceeding 10 and no modified groups. At the same time, the hydrophobicity and isoelectric point of the small peptides were analyzed using the peptide property calculator of Innovagen. Through analysis, it was found that most of the 37 small peptides had strong hydrophobicity. Small molecular peptides are more conducive to absorption and utilization, thus achieving a good lipid-lowering effect. The hydrophobicity of peptides also plays a positive role in lipid-lowering activity. Peptide segments with strong hydrophobicity are more likely to bind to bile acids. In addition, the isoelectric point of peptides is also one of the factors affecting lipid-lowering activity. A low isoelectric point is beneficial to the exertion of lipid-lowering activity, and most of the peptides identified in the present invention have a low isoelectric point.

[0064] Molecular docking: In the study of hypolipidemic activity and mechanism, pancreatic lipase and cholesterol esterase are two important enzymes. Pancreatic lipase produced by pancreatic acinar cells is responsible for hydrolyzing dietary triglycerides into diglycerides, monoglycerides, glycerol, and fatty acid anions. Inhibiting the activity of pancreatic lipase can effectively reduce the absorption efficiency of fat in the small intestine, thereby achieving the goal of lipid-lowering. Cholesterol esterase is a member of the α / β hydrolase family and is a bile salt-activated lipase that can catalyze the hydrolysis of dietary cholesterol esters into free cholesterol in the lumen of the small intestine. Inhibiting the activity of pancreatic cholesterol esterase can cause the serum cholesterol level in the diet to decrease at a reasonable rate, and it is considered an important target for treating cholesterol-related diseases. The docking software used is Autodock vina 1.1.2. Human pancreatic lipase (PDB ID: 1LPB) and human cholesterol esterase (PDB ID: 1F6W) are selected as receptor proteins, and their 3D structures are downloaded from the Protein Data Bank (PDB). The downloaded proteins are dehydrated and hydrogenated using ADT software. Molecular docking simulation studies are carried out on the identified 37 small peptides and the receptor proteins. By analyzing the binding energy between different peptides and the receptor proteins, peptides with potential high activity are screened. The 3D structure of the identified peptides is drawn by the software ChemBio3D. Among them, the hypolipidemic peptide with the sequence QIF has a relatively low binding energy in the molecular docking with the receptor proteins pancreatic lipase and cholesterol esterase. The docking results are shown in Table 1. This result theoretically indicates that the hypolipidemic peptide (QIF) has high pancreatic lipase inhibitory activity and cholesterol esterase inhibitory activity, that is, it has good hypolipidemic activity.

[0065] Table 1 Docking results of the hypolipidemic peptide QIF with pancreatic lipase and cholesterol esterase respectively

[0066]

[0067] To further study the molecular mechanism of the hypolipidemic peptide and the receptor protein, the binding sites and interaction forces of the hypolipidemic peptide with the two receptor proteins pancreatic lipase and cholesterol esterase were analyzed, and the results are as Figure 3 、 Figure 4 and Table 2 show. From Figure 3 and Table 2, it can be seen that the hypolipidemic peptide forms hydrogen bond interactions with amino acid residues such as Ser152, Phe77, His151, Tyr114, and Gly76 of pancreatic lipase, forms π-cation electrostatic interactions with Arg256, and forms hydrophobic interactions with amino acid residues such as Ile78, Ala259, Phe215, and Ala260; from Figure 4As can be seen from Table 2, the lipid-lowering peptide forms a traditional hydrogen bond connection with cholesterol esterase at Gly106, and forms van der Waals forces with amino acid residues such as Tyr105, Tyr125, Ile439, Val285, Leu282, Trp227, Ser194, Phe393, Ala195, His435, Phe324, Ala436, Ile323, Gly107 and Glu193, and forms hydrophobic interactions with amino acid residues such as Met281, Leu392, Met111, Ala108 and Leu110.

[0068] Table 2 Binding sites of lipid-lowering peptide QIF with pancreatic lipase and cholesterol esterase respectively

[0069]

[0070]

[0071] The above results show that the lipid-lowering peptide can interact with pancreatic lipase and cholesterol esterase through hydrogen bonds, electrostatic interactions, hydrophobic interactions and van der Waals forces. These interactions can lead to the formation of a complex between the lipid-lowering peptide and the receptor protein or affect the conformation of the enzyme, thereby affecting the normal physiological function of the receptor protein and further exerting lipid-lowering activity.

[0072] 1.3 Synthesis of lipid-lowering peptide

[0073] The lipid-lowering peptide was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. The purity of the polypeptide was >98%, which was used for subsequent research on its lipid-lowering activity.

[0074] Example 2 In vitro lipid-lowering activity of lipid-lowering peptide

[0075] The regulation of blood lipid levels is mainly achieved by controlling the levels of triglycerides and cholesterol in the body. Peptides can bind to bile salts, promote the degradation and metabolism of cholesterol, increase the excretion of steroids in fecal excreta, and thus reduce cholesterol absorption. In the treatment of hypercholesterolemia and obesity, the key metabolic enzymes related to lipid metabolism and cholesterol digestion, such as pancreatic lipase and cholesterol esterase, can be targeted for inhibition to achieve the treatment purpose. Traditional lipid-lowering drugs such as orlistat are developed based on this principle.

[0076] Taking the lipid-lowering peptide as a sample, the evaluation of bile salt binding ability, the determination of pancreatic lipase inhibitory activity and the determination of cholesterol esterase inhibitory activity were carried out respectively according to the aforementioned evaluation methods or test methods. Among them, the evaluation of bile salt binding ability selected the evaluation of sodium taurocholate binding ability as a representative.

[0077] The determination of sodium taurocholate binding ability, pancreatic lipase inhibitory activity and cholesterol esterase inhibitory activity is as followsFigure 5 As shown. From Figure 5 A, it can be seen that in the evaluation of the binding ability to sodium taurocholate, as the peptide concentration increases, the ability of the lipid-lowering peptide to bind sodium taurocholate gradually increases; from Figure 5 B, it can be seen that in terms of the inhibitory effect on pancreatic lipase, as the peptide concentration increases, the ability of the lipid-lowering peptide to inhibit the activity of pancreatic lipase gradually increases; from Figure 5 C, it can be seen that in terms of the inhibitory effect on cholesterol esterase, as the peptide concentration increases, the ability of the lipid-lowering peptide to inhibit the activity of cholesterol esterase gradually increases; from Figure 5 D, it can be seen that in terms of the inhibitory effect on pancreatic lipase, the activity of the lipid-lowering peptide (IC 50 : 0.013 mg / mL) is significantly better than that of the positive control orlistat (IC 50 : 0.017 mg / mL); in terms of the inhibitory effect on cholesterol esterase, the activity of the lipid-lowering peptide (IC 50 : 0.064 mg / mL) is close to that of the positive control orlistat (EC 50 = 0.051 mg / mL); in the evaluation of the binding ability to sodium taurocholate, the activity of the lipid-lowering peptide (EC 50 : 0.010 mg / mL) is significantly better than that of the positive control cholestyramine (EC 50 : 0.027 mg / mL). This indicates that the lipid-lowering peptide has strong bile salt binding ability, pancreatic lipase inhibitory effect and cholesterol esterase inhibitory effect.

[0078] Example 3 Effect of lipid-lowering peptide on lipid accumulation in 3T3-L1 preadipocytes

[0079] In recent years, 3T3-L1 preadipocytes have become one of the most typical cell lines for studying adipocyte differentiation, lipid accumulation and molecular mechanisms.

[0080] Cell culture and cell viability: 3T3-L1 cells were cultured in DMEM medium containing 10% FBS, and the cells were placed in a cell culture incubator at 5% CO 2 , 37 °C for culture. When the cells adhered and grew and the cell confluence reached more than 80%, they were digested and passaged with 0.25% trypsin solution or used for the next experiment. 100 μL of cell suspension (1×10 5 / mL) was inoculated into a 96-well plate. The control group and the experimental group were set up. After the cells adhered for 24 h, 100 μL of culture medium was added to the control group, and 100 μL of sample solutions with different concentrations was added to the experimental group. After culturing for 24 h, the old culture medium was discarded, and 100 μL of MTT (0.5 mg / mL) was added and incubated for 4 h. Finally, 100 μL of DMSO was added to each well, and the absorbance was measured at 490 nm. The formula for calculating cell viability:

[0081]

[0082] Where A t is the absorbance value of the experimental group, and A c is the absorbance value of the control group.

[0083] Cell induced differentiation: Inoculate the cells in a 24-well plate (3×10 4 cells / well), and first culture the cells with complete DMEM medium until the cells reach 90% confluence. Replace it with the primary induction differentiation medium and culture for 2 days. The primary induction differentiation medium is a complete medium containing 0.5 mM IBMX, 1 μM DEX, and 10 μg / mL insulin. Then culture with the secondary induction differentiation medium for 2 - 4 days, and change the medium every 2 days. The secondary induction differentiation medium is a complete medium containing 10 μg / mL insulin. Stop the differentiation until 80% of the cells have differentiated into mature adipocytes. During the induction differentiation, add samples with different concentrations to both the primary induction differentiation medium and the secondary induction differentiation medium.

[0084] First, through the MTT experiment, calculate the cell survival rate to determine the effect of the lipid-lowering peptide on the viability of 3T3-L1 cells.

[0085] Use the Oil Red O staining method to detect the effect of the lipid-lowering peptide on lipid accumulation in 3T3-L1 cells. After the induction differentiation of 3T3-L1 cells, a large number of lipid droplets will be generated, and the lipid droplets will be stained red by Oil Red O. Oil Red O staining method: After the induction differentiation is completed, aspirate the culture medium, wash the cells once with PBS buffer, fix the cells with 4% paraformaldehyde solution at room temperature for 10 min, and then wash the cells with PBS buffer. Stain according to the instructions of the Oil Red O kit, observe and take pictures under a microscope. After taking pictures, add isopropanol, shake for 5 min, and measure the absorbance value at a wavelength of 520 nm, which is recorded as the lipid content.

[0086] The results of cell survival rate, cell induced differentiation, and lipid accumulation in 3T3-L1 cells are as Figure 6 shown. From Figure 6 A, it can be seen that in the concentration range of 0.005 - 0.05 mg / mL, the lipid-lowering peptide has a promoting effect on the growth of 3T3-L1 cells. When the concentration is 0.05 mg / mL, the lipid-lowering peptide has no obvious negative effect on the growth of 3T3-L1 cells. The positive control simvastatin only set experiments at low concentrations (0.005 mg / mL and 0.01 mg / mL) because the positive control simvastatin at higher concentrations inhibits cell growth. From Figure 6 B, it can be seen that compared with the model group, the number of lipid droplets stained after the differentiation of cells added with the lipid-lowering peptide is significantly reduced, indicating that the lipid-lowering peptide can effectively inhibit the adipogenic differentiation of 3T3 cells. From Figure 6As can be seen from C, quantitative analysis found that as the concentration of the lipid-lowering peptide increased, the lipid content in adipocytes containing the lipid-lowering peptide also significantly decreased gradually. At a concentration of 0.05 mg / mL, the lipid content of the cells treated with the lipid-lowering peptide decreased by 18.28% compared with the model group.

[0087] Example 4 Effect of Lipid-lowering Peptide on Lipoprotein Levels in Adipocytes

[0088] One of the main means of regulating high cholesterol levels is to reduce the level of low-density lipoprotein (LDL) while maintaining the level of high-density lipoprotein (HDL). Low-density lipoprotein can carry cholesterol into tissues, bind to biological membranes, or be converted into steroids. Excessive low-density lipoprotein will increase the cholesterol level in the circulation and accumulate in vascular tissues, leading to a series of cardiovascular problems. High-density lipoprotein, on the other hand, plays an important role in the reverse cholesterol transport mechanism. This mechanism can remove excess cholesterol through macrophages in the arterial wall and transport it to the liver for excretion, achieving the dynamic balance of total cholesterol. Therefore, maintaining an appropriate high-density lipoprotein cholesterol level is crucial for preventing dyslipidemia.

[0089] After the cell induction and differentiation were completed, the cell precipitate was collected, washed with PBS buffer, and then ultrasonically disrupted. Detection was carried out according to the instructions of the high-density lipoprotein and low-density lipoprotein kits. The effects of the lipid-lowering peptide on the high-density lipoprotein and low-density lipoprotein levels in 3T3-L1 cells are as Figure 7 shown. As can be seen from Figure 7 A, compared with the model group, the low-density lipoprotein level of the cells treated with the lipid-lowering peptide was significantly reduced, and as the concentration of the lipid-lowering peptide increased, the low-density lipoprotein level decreased more; as can be seen from Figure 7 B, compared with the model group, the high-density lipoprotein level of the cells treated with the lipid-lowering peptide increased, and as the concentration of the lipid-lowering peptide increased, the high-density lipoprotein level increased more. At a concentration of 0.05 mg / mL, the high-density lipoprotein level of the cells treated with the lipid-lowering peptide increased by 45.09% and the low-density lipoprotein level decreased by 49.10% compared with the model group.

[0090] In summary, the lipid-lowering peptide can play a lipid-lowering role through in vitro bile salt binding, pancreatic lipase inhibition, and cholesterol esterase inhibition, and can exert lipid-lowering activity by regulating lipid and lipoprotein cholesterol levels in 3T3-L1 cells.

[0091] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill 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 implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of a lipid-lowering peptide in the preparation of a lipid-lowering drug, characterized in that, the sequence of the lipid-lowering peptide is: QIF.

2. The use of the lipid-lowering peptide according to claim 1, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for reducing the lipid content in cells.

3. The use of the lipid-lowering peptide according to claim 1, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for reducing the content of low-density lipoprotein in cells and increasing the content of high-density lipoprotein in cells.

4. The use of the lipid-lowering peptide according to claim 1, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for promoting cholesterol degradation and metabolism.

5. The use of the lipid-lowering peptide according to claim 4, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for binding to bile salts.

6. The use of the lipid-lowering peptide according to claim 1, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for inhibiting the activity of pancreatic lipase.

7. The use of the lipid-lowering peptide according to claim 1, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for inhibiting the activity of cholesterol esterase.

8. The use of the lipid-lowering peptide according to claim 1, characterized in that, the use of the lipid-lowering peptide in the preparation of a drug for inhibiting adipogenic differentiation of cells.

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