A garlic peptide with lipid-lowering function and its preparation method and application

Abstract

The present invention relates to the field of polypeptide technology, and in particular to a garlic peptide with lipid-lowering function, its preparation method, and application. The present invention provides a garlic peptide with lipid-lowering function, wherein the garlic peptide is one or more of FL7, PF7, AT8, GG8, or AR9. The garlic peptide sequence obtained by the present invention can effectively inhibit pancreatic lipase activity while significantly reducing the levels of total triglycerides and total cholesterol in oleic acid-induced hyperlipidemia HepG2 cells. The garlic peptide sequence of the present invention is naturally non-toxic, highly stable, and has a clear target of action, meeting the requirements of pharmaceutical formulation development. It can be used as a lipid-functional molecule in special medical foods, health products, and drugs for the treatment or auxiliary treatment of dyslipidemia.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a garlic peptide with lipid-lowering function, and a preparation method and application thereof. Background Art

[0002] Dyslipidemia, commonly known as hyperlipidemia, is a very common condition in modern times. It is a major cause of atherosclerosis and an independent risk factor for coronary heart disease and ischemic stroke. Dyslipidemia is generally divided into four categories based on clinical experience: hypercholesterolemia (TC), hypertriglyceridemia (TG), combined hyperlipidemia, and low high-density lipoprotein (LDL).

[0003] Dyslipidemia is typically manifested by elevated levels of TC, TG, and LDL cholesterol, and in severe cases, can also lead to decreased high-density lipoprotein (HDL). Lipid-lowering medications can treat dyslipidemia by improving levels of TC and TG, but existing Western medications, such as pancreatic lipase inhibitors (orlistat) and HMGCR inhibitors (statins), have toxic side effects.

[0004] Food-derived bioactive peptides have the advantages of easy absorption, high bioavailability, low allergenicity, and stable properties. Therefore, it is particularly important to develop new, efficient, and safe food-derived bioactive peptide lipid-lowering drugs. Summary of the Invention

[0005] The present invention uses garlic as the main raw material, and designs a new preparation process with the pancreatic lipase inhibition rate and the reduction of lipid content in the oleic acid-induced high-fat HepG2 cell model as indicators. The prepared garlic lipid-lowering peptide can be used for the treatment or auxiliary treatment of dyslipidemia.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a garlic peptide with lipid-lowering function, wherein the garlic peptide is one of FL7, PF7, AT8, GG8 or AR9;

[0008] The amino acid sequence of the FL7 is: FDPLAQL, the amino acid sequence of the PF7 is: PRPNEYF, the amino acid sequence of the AT8 is: AGWLFVST, the amino acid sequence of the GG8 is: GWLFVSTG, and the amino acid sequence of the AR9 is: AYDVFGSPR.

[0009] The present invention also provides a method for preparing the garlic peptide having lipid-lowering function, comprising the following steps:

[0010] (1) mixing garlic and water, homogenizing the mixture, and filtering to obtain a filtrate; mixing the filtrate with ammonium sulfate, allowing the mixture to stand, and centrifuging to obtain a precipitate;

[0011] (2) mixing the precipitate of step (1) with phosphate buffer for redissolution, dialyzing, and freeze-drying to obtain garlic crude protein powder, and preparing a garlic crude protein solution;

[0012] (3) mixing the garlic crude protein solution of step (2) with alkaline protease for enzymatic hydrolysis, centrifuging, and collecting the supernatant as the enzymatic hydrolysis product;

[0013] (4) The enzymatic hydrolysis product obtained in step (3) is ultrafiltered, and the components with molecular weight <3.5 kDa are taken for purification, identification, and virtual screening.

[0014] Preferably, the mass ratio of the garlic to water in step (1) is 1:4-8; and the saturation degree of the ammonium sulfate is 75-85%.

[0015] Preferably, the centrifugal speed in step (1) is 7000-9000 r / min, and the time is 4-6 min; the standing temperature is 3-5° C., and the time is 10-16 h.

[0016] Preferably, the garlic crude protein solution in step (2) uses water as solvent; the mass concentration of the garlic crude protein solution is 0.3-0.7%; and the pH of the garlic crude protein solution is 9-10.

[0017] Preferably, the amount of alkaline protease added in step (3) is 8000-12000 U / g; the temperature of the enzymatic hydrolysis is 40-50° C., and the time is 2-4 hours; the speed of the centrifugation is 7000-9000 rpm, and the time is 15-25 minutes.

[0018] Preferably, the purification method in step (3) is:

[0019] Mix the components with molecular weight <3.5KDa with water to obtain the solution to be purified; load the solution to be purified into a 1.6cm×55cm chromatography column, and after complete natural sedimentation, balance it with ultrapure water for 1-2 hours; the single column loading volume is 2mL, the elution flow rate is 1mL / min, the elution solution is ultrapure water, collect one tube every 10 minutes, collect the components of 50-100 minutes, and obtain the purified polypeptide sample.

[0020] Preferably, step (3) is identified using MALDI-TOF mass spectrometry, specifically comprising the following steps:

[0021] Prepare a purified peptide sample solution with a final concentration of 2-3 μg / μL using 0.1% trichloroacetic acid as solvent;

[0022] Primary mass spectrometry analysis conditions: linear positive ion mode, detection range of 700-3500 Da, nitrogen laser, wavelength of 340-360 nm, laser frequency of 450-550 Hz, energy range of 20%-50%, and peptide standard II for calibration;

[0023] Secondary mass spectrometry analysis conditions: Lift secondary mass spectrometry analysis method, secondary detection range determined by primary parent ion, secondary detection range of 200-1600 Da, laser energy range of 18%-80%;

[0024] The analysis software FlexAnalysis 3.4 was used for automatic peak marking, and Mascot was used to search the library of protein primary and secondary mass spectrometry information to obtain the best matching data for identification of peptides.

[0025] Preferably, the virtual screening method in step (3) is as follows: the activity probability of the identified peptides is predicted by the online database PeptideRanker, and a score of >0.5 is used as a threshold to screen out peptide sequences with a higher probability of being active; the physicochemical properties and toxicity of the peptide sequences are predicted using Pepdraw, PepCalc and ToxinPred; and peptide sequences with an activity score >0.5 are screened for peptide sequences that inhibit pancreatic lipase activity using molecular docking.

[0026] The present invention also provides the garlic peptide with lipid-lowering function or the use of the garlic peptide with lipid-lowering function prepared by the method in the preparation of lipid-lowering drugs.

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

[0028] The garlic peptide obtained by the present invention can effectively inhibit pancreatic lipase activity and significantly reduce total triglyceride and total cholesterol levels in oleic acid-induced hyperlipidemia HepG2 cells. Furthermore, the garlic peptide sequence described in the present invention is naturally non-toxic, highly stable, and has a clear target, meeting the requirements for pharmaceutical formulation development. It can be used as a lipid-functional molecule in special medical foods, health products, and pharmaceuticals for the treatment or adjunctive treatment of dyslipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0030] Figure 1 This is the pancreatic lipase inhibitory effect of the ultrafiltration fraction of Example 1 of the present invention.

[0031] Figure 2 This is the elution curve of Sephadex G-25 in Example 1 of the present invention.

[0032] Figure 3 This is the HPLC result of FL7.

[0033] Figure 4 This is the MS result of FL7.

[0034] Figure 5 This is the HPLC result of PF7.

[0035] Figure 6 This is the MS result of PF7.

[0036] Figure 7 This is the HPLC result of AT8.

[0037] Figure 8 This is the MS result of AT8.

[0038] Figure 9 This is the HPLC result of GG8.

[0039] Figure 10 This is the MS result of GG8.

[0040] Figure 11 This is the HPLC result of AR9.

[0041] Figure 12 This is the MS result of AR9.

[0042] Figure 13 It is the IC50 of the artificially synthesized peptides FL7, PF7, AT8, GG8, and AR9 of Example 1 of the present invention against pancreatic lipase.

[0043] Figure 14 This is the effect of Example 1 of the present invention on total triglycerides in the high-fat HepG2 cell model induced by oleic acid.

[0044] The blank group was HepG2 cells cultured without oleic acid; the model group was cells induced with 0.5 mM oleic acid; the positive control group (OR) was cells cultured with orlistat and oleic acid; and the sample group was cells cultured with peptides of different sequences and oleic acid.

[0045] Figure 15 This is the effect of Example 1 of the present invention on total cholesterol in the high-fat HepG2 cell model induced by oleic acid.

[0046] The blank group was HepG2 cells cultured without oleic acid; the model group was cells induced with 0.5 mM oleic acid; the positive control group (OR) was cells cultured with orlistat and oleic acid; and the sample group was cells cultured with peptides of different sequences and oleic acid. DETAILED DESCRIPTION

[0047] The present invention provides a garlic peptide with lipid-lowering function, wherein the garlic peptide is one of FL7, PF7, AT8, GG8 or AR9;

[0048] The amino acid sequence of the FL7 is: FDPLAQL, the amino acid sequence of the PF7 is: PRPNEYF, the amino acid sequence of the AT8 is: AGWLFVST, the amino acid sequence of the GG8 is: GWLFVSTG, and the amino acid sequence of the AR9 is: AYDVFGSPR.

[0049] GG8 is preferred.

[0050] The present invention also provides a method for preparing the garlic peptide having lipid-lowering function, comprising the following steps:

[0051] (1) mixing garlic and water, homogenizing the mixture, and filtering to obtain a filtrate; mixing the filtrate with saturated ammonium sulfate, allowing the mixture to stand, and centrifuging to obtain a precipitate;

[0052] (2) mixing the precipitate of step (1) with phosphate buffer for redissolution, dialyzing, and freeze-drying to obtain garlic crude protein powder, and preparing a garlic crude protein solution;

[0053] (3) mixing the garlic crude protein solution of step (2) with alkaline protease for enzymatic hydrolysis, centrifuging, and collecting the supernatant as the enzymatic hydrolysis product;

[0054] (4) The enzymatic hydrolysis product obtained in step (3) is ultrafiltered, and the components with molecular weight <3.5 kDa are taken for purification, identification, and virtual screening.

[0055] In the present invention, the mass ratio of the garlic to water in step (1) is 1:4-8; preferably 1:5-7; more preferably 1:6.

[0056] The saturation degree of the saturated ammonium sulfate is 75-85%, preferably 77-83%, more preferably 79-81%, and more preferably 80%.

[0057] In the present invention, the centrifugal speed in step (1) is 7000-9000 r / min, and the time is 4-6 min; preferably, the speed is 7200-8800 r / min, and the time is 5 min; further preferably, the speed is 7400-8600 r / min, and the time is 5 min; more preferably, the speed is 8000 r / min, and the time is 5 min.

[0058] In the present invention, the standing temperature in step (1) is 3-5°C and the standing time is 10-16h; preferably, the standing temperature is 4°C and the standing time is 10-16h; more preferably, the standing temperature is 4°C and the standing time is 12-14h; more preferably, the standing temperature is 4°C and the standing time is 13h.

[0059] In the present invention, the dialysis time in step (2) is 2 to 4 days, preferably 3 days.

[0060] In the present invention, the garlic crude protein solution in step (2) uses water as solvent;

[0061] In the present invention, the mass concentration of the garlic crude protein solution in step (2) is 0.3-0.7%, preferably 0.4-0.6%, and more preferably 0.5%.

[0062] The pH of the garlic crude protein solution is 9-10, preferably 9.5.

[0063] In the present invention, the amount of alkaline protease added in step (3) is 8000-12000 U / g; preferably 9000-11000 U / g; more preferably 10000 U / g.

[0064] In the present invention, the enzymatic hydrolysis temperature in step (3) is 40-50°C and the time is 2-4h; preferably, the enzymatic hydrolysis temperature is 42-48°C and the time is 2-4h; more preferably, the enzymatic hydrolysis temperature is 44-46°C and the time is 2-4h; more preferably, the enzymatic hydrolysis temperature is 45°C and the time is 3h.

[0065] In the present invention, the centrifugal speed in step (3) is 7000-9000 rpm, and the time is 15-25 min; preferably, the centrifugal speed is 7200-8800 rpm, and the time is 17-23 min; further preferably, the centrifugal speed is 7400-8600 rpm, and the time is 19-21 min; more preferably, the centrifugal speed is 8000 rpm, and the time is 20 min.

[0066] In the present invention, the purification method in step (3) is:

[0067] Mix the components with molecular weight <3.5KDa with water to obtain the solution to be purified; load the solution to be purified into a 1.6cm×55cm chromatography column, and after complete natural sedimentation, balance it with ultrapure water for 1-2 hours; the single column loading volume is 2mL, the elution flow rate is 1mL / min, the elution solution is ultrapure water, collect one tube every 10 minutes, collect the components of 50-100 minutes, and obtain the purified polypeptide sample.

[0068] In the present invention, after natural sedimentation is complete, ultrapure water is used for 2 hours of equilibration.

[0069] In the present invention, step (3) is identified using MALDI-TOF mass spectrometry, which specifically includes the following steps:

[0070] Prepare a purified peptide sample solution with a final concentration of 2-3 μg / μL using 0.1% TFA as solvent;

[0071] Primary mass spectrometry analysis conditions: linear positive ion mode, detection range of 700-3500 Da, nitrogen laser, wavelength of 340-360 nm, laser frequency of 450-550 Hz, energy range of 20%-50%, and peptide standard II for calibration;

[0072] Secondary mass spectrometry analysis conditions: Lift secondary mass spectrometry analysis method, secondary detection range determined by primary parent ion, secondary detection range of 200-1600 Da, laser energy range of 18%-80%;

[0073] The analysis software FlexAnalysis 3.4 was used for automatic peak marking, and Mascot was used to search the library of protein primary and secondary mass spectrometry information to obtain the best matching data for identification of peptides.

[0074] In the present invention, the virtual screening method in step (3) is as follows: the activity probability of the identified peptides is predicted by the online database Peptide Ranker, and a score of >0.5 is used as a threshold to screen out peptide sequences with a higher probability of being active; the physicochemical properties and toxicity of the peptide sequences are predicted by using Pepdraw, PepCalc and ToxinPred; and the peptide sequences with an activity score >0.5 are screened for peptide sequences that inhibit pancreatic lipase activity by molecular docking.

[0075] In the present invention, the primary mass spectrometry analysis conditions are: linear positive ion mode, a detection range of 700 to 3500 Da, a nitrogen laser, a wavelength of 342 to 358 nm, a laser frequency of 452 to 548 Hz, an energy range of 20% to 50%, and a polypeptide standard II for calibration; further preferably, the primary mass spectrometry analysis conditions are: linear positive ion mode, a detection range of 700 to 3500 Da, a nitrogen laser, a wavelength of 355 nm, a laser frequency of 500 Hz, an energy range of 20% to 50%, and a polypeptide standard II for calibration.

[0076] The present invention also provides the garlic peptide with lipid-lowering function or the use of the garlic peptide with lipid-lowering function prepared by the method in the preparation of lipid-lowering drugs.

[0077] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0078] Example 1

[0079] A method for preparing garlic peptide with lipid-lowering function, comprising the following steps:

[0080] 1) Raw material pretreatment: Peel the fresh garlic cloves and place the garlic and water in a wall-breaking machine at a mass ratio of 1:5, crush and homogenize, and filter. Add 80% saturated ammonium sulfate (25°C) to the filtrate and place it in a 4°C refrigerator for 12 hours. Centrifuge the filtrate at 8000 rpm for 5 minutes, discard the supernatant, and reconstitute the precipitate with phosphate buffer (the phosphate buffer is added in no specific ratio; the purpose is to reconstitute the crude garlic protein for easy transfer. It is then freeze-dried in a vacuum dryer for easy storage). Place the precipitate in a 3KDa dialysis bag and dialyze for 3 days. The dialysate is freeze-dried in a freeze dryer (vacuum: 10P, cold trap temperature -60°C, freeze-drying time: 3 days) to obtain crude garlic protein powder.

[0081] 2) Enzymatic Hydrolysis of Garlic Crude Protein: A 0.5% garlic crude protein solution was prepared using grade tertiary water, the pH adjusted to 9.5, and enzymatic hydrolysis was performed at 45°C for 3 h (using 10,000 U / g of Alcalase). After completion, the solution was inactivated in a boiling water bath for 10 min. After cooling, the solution was centrifuged at 8,000 rpm for 20 min, and the supernatant was collected as the enzymatic hydrolysis product.

[0082] 3) Isolation, Purification, and Identification of Garlic Lipid-Lowering Peptides: The enzymatic hydrolysates were separated using ultrafiltration, and the pancreatic lipase inhibition rates of the different fractions were determined. The highly active fractions were further purified and separated using dextran gel, and the peptide sequences were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The specific methods are as follows:

[0083] ① Ultrafiltration separation: Distilled water was added to the enzymatic hydrolysate, and the sample was ultrafiltered using a FlowMem0015 ultrafiltration flat membrane pilot test device. Using a 3.5 kDa membrane, fractions were separated into those >3.5 kDa and <3.5 kDa, and the pancreatic lipase inhibition rates of the different molecular weight fractions were determined. Figure 1 The results showed that the pancreatic lipase inhibitory activity of the <3.5KDa fraction was significantly stronger than that of the >3.5KDa fraction.

[0084] The specific experimental process for determining the pancreatic lipase inhibition rate is as follows:

[0085] Prepare a 50 mM solution of p-nitroacetate (PNPA) in dimethyl sulfoxide (DMSO), then dilute to 10 mM with distilled water as the substrate solution. Prepare a 10 mg / mL solution of porcine pancreatic lipase, centrifuge at 12,000 rpm for 5 minutes, and collect the supernatant.

[0086] A reaction system was prepared in a 96-well plate, including 20 μL of ultrafiltered sample, 20 μL of porcine pancreatic lipase solution, 20 μL of PNPA substrate solution, and 140 μL of Tris-HCl buffer (pH = 7.4). After adding the substrate to initiate the reaction for 10 minutes, the absorbance at 405 nm was immediately measured, and the pancreatic lipase inhibition rate was calculated according to the following formula 1:

[0087]

[0088] Where A1 is the absorbance of the enzyme-containing solution after adding peptide, A2 is the absorbance of the sample solution without enzyme, and A3 is the absorbance of the solution without peptide (maximum activity of the enzyme).

[0089] ②Sephadex chromatography: Dissolve the <3.5KDa component in ultrapure water to make a 25mg / mL solution, load the filler into a 1.6cm×55cm chromatography column, and after natural sedimentation, balance with ultrapure water for 1-2h. The single column loading volume is 2mL, the elution flow rate is 1mL / min, the elution solution is ultrapure water, collect one tube every 10min, and measure the absorbance at 220nm. The elution curve is shown in Figure 2. Figure 2 , according to the peak time, it can be roughly divided into two components F1 and F2.

[0090] ③ MALDI-TOF MS peptide sequence identification: Prepare a 10 mg / mL matrix solution of α-cyano-4-hydroxycinnamic acid. Take 3 mg of peptide sample and dissolve it in 0.1% TFA to a final protein concentration of 3 μg / μL. Primary mass spectrometry analysis conditions: Linear positive ion mode, detection range of 3500 Da, nitrogen laser, wavelength of 355 nm, laser frequency of 500 Hz, energy range of 50%, and peptide standard II for calibration. Secondary mass spectrometry analysis conditions: Lift secondary mass spectrometry method, secondary detection range determined by primary precursor ion (m / z), secondary detection range of 200-1600 Da, laser energy range of 18%-80%. FlexAnalysis 3.4 analysis software was used for automatic peak processing. Mascot was used to search the primary and secondary mass spectrometry databases of the protein, and the best matching data was used for identification of the peptide.

[0091] 4) Virtual Screening of Garlic Lipid-Lowering Peptides: The identified peptide sequences were used to predict activity probabilities using the online database Peptide Ranker. Using a score > 0.5 as a threshold, peptide sequences with a high probability of activity were screened. The physicochemical properties and toxicity of these peptide sequences were predicted using Pepdraw, PepCalc, and ToxinPred. The results are shown in Table 1, indicating that none of these peptide sequences were toxic. Peptide sequences with activity scores > 0.5 were then used for molecular docking to screen for peptide sequences that inhibit pancreatic lipase activity.

[0092] The present invention uses AutoDock Vina to perform molecular docking, obtains the crystal structure of the receptor from the protein structure database PDB (RCSB PDB: Homepage), selects human pancreatic lipase as the docking receptor, and its docking binding energy and hydrogen bond interaction are shown in Table 2. The binding energy of most peptide sequences is lower than -5.0Kcal / mol, indicating that they can form a stable conformation with pancreatic lipase. From the perspective of hydrogen bond formation, orlistat generates 6 hydrogen bonds when docked with pancreatic lipase, and can form hydrogen bonds with SER152 and HIS263 in the catalytic triad, resulting in a stable inhibitory effect. While the number of hydrogen bonds generated by other active peptides when docked with pancreatic lipase is less than that of orlistat, GG8, AT8, PF7, FT11, GR13, SW12 and LD16 can all form hydrogen bonds with some amino acid residues in the catalytic triad.

[0093] In summary, the molecular docking results showed that the five peptides with shorter sequences, FL7 (FDPLAQL), ​​PF7 (PRPNEYF), AT8 (AGWLFVST), GG8 (GWLFVSTG), and AR9 (AYDVFGSPR), bound more stably to the catalytic site of pancreatic lipase and could effectively inhibit its activity.

[0094] Table 1 Activity scores of peptide sequences and predictions of physicochemical properties from online databases

[0095]

[0096]

[0097] As shown in SEQ ID NOs: 1-10.

[0098] Table 2 Prediction of binding of AutoDock Vina docking peptides to receptors 1LPB and 1HW9

[0099]

[0100]

[0101] 5) Study on the lipid-lowering function of garlic peptides: Based on the results of step 4), the peptide sequences FL7, PF7, AT8, GG8, and AR9 were synthesized by artificial solid phase synthesis, and the purity and molecular weight of the synthesized peptides were identified by high performance liquid chromatography and mass spectrometry. Figures 3 to 12 The results show that the purity of the synthesized peptides is ≥95% and the molecular weight deviation is small. The results of the pancreatic lipase inhibitory activity are shown in Figure 13 .

[0102] Furthermore, the effects of five synthetic peptide sequences on triglyceride and cholesterol in HepG2 cells induced by oleic acid (OA) were determined.

[0103] Logarithmic phase HepG2 cells were digested and diluted to 1.5×10 6 cell / mL, 1 mL was inoculated into a 6-well plate in an incubator and cultured for 24 h. After the cells attached to the wall, DMEM medium containing different concentrations of synthetic peptide samples and oleic acid modeling solution was added and cultured for 1 day.

[0104] The oleic acid modeling solution was prepared by dissolving a certain amount of oleic acid in 0.1 mM NaOH at 70°C to create a 100 mM oleic acid solution. This solution was then slowly added dropwise to a PBS solution containing 10% bovine serum albumin (BSA) to create a 5 mM oleic acid stock solution. The solution was shaken thoroughly to eliminate oil droplets. The solution was filtered through a microporous membrane in a clean bench, aliquoted, and stored in a -20°C refrigerator. Upon use, it was diluted to 0.5 mM in DMEM for culture as the modeling agent.

[0105] After 24 hours of intervention, the old culture medium was aspirated and the cells were washed twice with PBS. 200 μL of RIPA cell lysis buffer was added to each well and the cells were lysed on ice for 30 minutes. The TC and TG contents in the lysate were determined according to the kit instructions. The changes in total triglyceride and total cholesterol in the cells were shown in Figure 2. Figure 14 and Figure 15 As shown in the figure, * indicates significant difference between the model group and the blank group (*P<0.05; ***P<0.01), # indicates significant difference between the drug-treated group and the model group (#P<0.05; ###P<0.01) (the blank group was serum-free DMEM culture medium, and the model group was serum-free DMEM culture medium containing 0.5 mM oleic acid modeling solution).

[0106] The experimental results showed that garlic peptides FL7, PF7, AT8, GG8, and AR9 can effectively inhibit pancreatic lipase activity, and their inhibitory intensity is very similar to the results of molecular docking simulation. At the same time, by evaluating the effects on the total triglyceride and total cholesterol levels of the oleic acid-induced high-fat HepG2 cell model, garlic lipid-lowering peptides can significantly reduce the intracellular lipid accumulation caused by oleic acid.

[0107] In summary, garlic peptides FL7, PF7, AT8, GG8, and AR9 can effectively inhibit pancreatic lipase activity and significantly improve total triglyceride and total cholesterol levels in oleic acid-induced cell models. They are safe and non-toxic, and can be used in the development of pharmaceutical preparations or health foods for the treatment or adjuvant treatment of dyslipidemia. Based on the pancreatic lipase inhibition rate and cell experiments, GG8 has the strongest potential lipid-lowering effect.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A garlic peptide with lipid-lowering function, characterized in that: The garlic peptide is one of FL7, PF7, AT8, GG8 or AR9; The amino acid sequence of the FL7 is: FDPLAQL, the amino acid sequence of the PF7 is: PRPNEYF, the amino acid sequence of the AT8 is: AGWLFVST, the amino acid sequence of the GG8 is: GWLFVSTG, and the amino acid sequence of the AR9 is: AYDVFGSPR.

2. The method for preparing the garlic peptide with lipid-lowering function according to claim 1, characterized in that: The steps include: (1) mixing garlic and water, homogenizing the mixture, and filtering to obtain a filtrate; mixing the filtrate with ammonium sulfate, allowing the mixture to stand, and centrifuging to obtain a precipitate; (2) mixing the precipitate of step (1) with phosphate buffer for redissolution, dialyzing, and freeze-drying to obtain garlic crude protein powder, and preparing a garlic crude protein solution; (3) mixing the garlic crude protein solution of step (2) with alkaline protease for enzymatic hydrolysis, centrifuging, and collecting the supernatant as the enzymatic hydrolysis product; (4) The enzymatic hydrolysis product obtained in step (3) is ultrafiltered, and the components with molecular weight <3.5 kDa are taken for purification, identification, and virtual screening.

3. The method according to claim 2, characterized in that The mass ratio of the garlic to water in step (1) is 1:4-8; and the saturation degree of the ammonium sulfate is 75-85%.

4. The method according to claim 2, characterized in that The centrifugal speed in step (1) is 7000-9000 r / min, and the time is 4-6 min; the standing temperature is 3-5° C., and the time is 10-16 h.

5. The method according to claim 2, characterized in that The garlic crude protein solution in step (2) uses water as solvent; the mass concentration of the garlic crude protein solution is 0.3-0.7%; and the pH value of the garlic crude protein solution is 9-10.

6. The method according to claim 2, characterized in that In step (3), the amount of alkaline protease added is 8000-12000 U / g; the temperature of the enzymatic hydrolysis is 40-50° C., and the time is 2-4 hours; the speed of the centrifugation is 7000-9000 rpm, and the time is 15-25 minutes.

7. The method according to claim 2, characterized in that The purification method in step (3) is: Mix the components with molecular weight <3.5KDa with water to obtain the solution to be purified; load the solution to be purified into a 1.6cm×55cm chromatography column, and after complete natural sedimentation, balance it with ultrapure water for 1-2 hours; the single column loading volume is 2mL, the elution flow rate is 1mL / min, the elution solution is ultrapure water, collect one tube every 10 minutes, collect the components of 50-100 minutes, and obtain the purified polypeptide sample.

8. The method according to claim 7, characterized in that Step (3) uses MALDI-TOF mass spectrometry for identification, specifically comprising the following steps: The purified peptide sample solution was prepared with 0.1% trifluoroacetic acid as solvent to a final concentration of 2-3 μg / μL; Primary mass spectrometry analysis conditions: linear positive ion mode, detection range of 700-3500 Da, nitrogen laser, wavelength of 340-360 nm, laser frequency of 450-550 Hz, energy range of 20%-50%, and peptide standard II for calibration; Secondary mass spectrometry analysis conditions: Lift secondary mass spectrometry analysis method, secondary detection range determined by primary parent ion, secondary detection range of 200-1600 Da, laser energy range of 18%-80%; The analysis software FlexAnalysis 3.4 was used for automatic peak marking, and Mascot was used to search the library of protein primary and secondary mass spectrometry information to obtain the best matching data for identification of peptides.

9. The method according to claim 8, characterized in that The virtual screening method in step (3) is as follows: the activity probability of the identified peptides is predicted by the online database Peptide Ranker, and the active peptide sequences are screened with a score of >0.5 as a threshold; the physicochemical properties and toxicity of the peptide sequences are predicted using Pepdraw, PepCalc and ToxinPred; and the peptide sequences with an activity score >0.5 are screened for peptide sequences that inhibit pancreatic lipase activity using molecular docking.

10. Use of the garlic peptide with lipid-lowering function according to claim 1 or the garlic peptide with lipid-lowering function prepared by the method according to any one of claims 2 to 9 in the preparation of lipid-lowering drugs.

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