Golden imperial chrysanthemum oligopeptide and 9 peptide with effects of reducing blood fat and losing weight as well as preparation method and application of golden imperial chrysanthemum oligopeptide and 9 peptide

Abstract

The invention belongs to the technical field of medical technology and deep processing of vegetable protein polypeptides, and discloses golden chrysanthemum oligopeptide with blood fat reducing and weight losing effects, which has pancreatic lipase inhibition activity and cellular level blood fat reducing activity. The oligopeptide has pancreatic lipase inhibitory activity, and also has an inhibitory effect on fatty acid-induced accumulation of HepG2 cell lipid content. A plurality of polypeptide sequences are identified in the golden chrysanthemum oligopeptide through a mass spectrum method, and the sequence of the 9 peptide with the highest confidence coefficient is AGDTHLGGE and also has good pancreatic lipase inhibitory activity. The production process of the plant protein is green and environment-friendly, and the product is good in safety and has good pancreatic lipase inhibition activity and cellular level lipid-lowering activity. Therefore, the golden chrysanthemum petal protein oligopeptide and 9 peptide disclosed by the invention have good effects of reducing blood fat and controlling body weight, and have application prospects in the aspects of special medical foods, health foods, functional foods, medicines and common foods.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and deep processing technology of plant protein polypeptides, and in particular relates to the preparation and application of plant proteins and polypeptides, and in particular to a golden chrysanthemum oligopeptide and a 9-peptide with lipid-lowering and weight-loss effects, and a preparation method and application thereof. Background Art

[0002] In recent years, with the improvement of living standards, hyperlipidemia has become a common clinical symptom. Hyperlipidemia is a disease of abnormal lipid metabolism and one of the causes of various cardiovascular diseases. It is estimated that by 2030, the number of cardiovascular and cerebrovascular patients in my country will continue to increase to 170 million, and there is a trend of younger age. Hyperlipidemia is also a major complication of diseases such as obesity, diabetes, and chronic kidney disease. Currently, the lipid-lowering drugs widely used in clinical practice mainly achieve the purpose of lipid-lowering by reducing the intake of cholesterol in the diet, inhibiting the synthesis process of cholesterol in the human body or accelerating its decomposition rate, and reducing the concentration of lipids in plasma. Statins are common drugs in the mechanism of inhibiting cholesterol synthesis, but long-term use has adverse reactions such as muscle weakness and muscle pain. Therefore, it is of great research significance and market value to find lipid-lowering drugs or functional foods with low toxic side effects.

[0003] Pancreatic lipase digests fat in the human body. Lipase inhibitors can effectively inhibit the hydrolysis of fat by lipase, thereby preventing the body from absorbing fat, and have the effects of controlling blood lipids and treating obesity. Orlistat, an existing drug on the market, is a lipase inhibitor that can inhibit triglyceride absorption, reduce calorie intake, control body weight, and reduce the risk of health problems caused by obesity.

[0004] Chrysanthemum scabra is a perennial herb belonging to the genus Chrysanthemum of the Asteraceae family. Its unique ornamental value, aromatic and sweet taste, and good medical effects make it very popular. There are many reports on small and medium-molecule active substances in Chrysanthemum scabra, but there are relatively few reports on protein and polypeptide substances. The applicant has conducted in-depth research on Chrysanthemum scabra for many years and has found that Chrysanthemum scabra peptide has lipid-lowering and antioxidant effects. In further research, it was found that after process improvement, Chrysanthemum scabra peptide also has good lipid-lowering activity.

[0005] Through searching, the following public documents related to the patent application of the present invention are found:

[0006] Patent 1: Guo Na, Sun Hua, et al., a golden chrysanthemum protein polypeptide and its preparation and application. Application number 202110458702.4. Disclosed is a method of using golden chrysanthemum powder as raw material, extracting protein therein by alkali dissolution and acid precipitation method, and then hydrolyzing chrysanthemum protein by protease to obtain low molecular weight chrysanthemum polypeptide, which has hypoglycemic and antioxidant activity. The present invention is different from the above invention in several ways. First, the present invention found that golden chrysanthemum polypeptide has good lipid-lowering activity, while the above invention is hypoglycemic activity; second, there are several differences in the preparation process of the present invention. The present invention adds "defatting" and "dialysis" steps in the protein preparation process, and the appearance and nitrogen content of the obtained golden chrysanthemum protein are significantly improved and increased.

[0007] By comparison, previous reports did not disclose the lipid-lowering activity. Therefore, the present invention is essentially different from the above patents. First, the lipid-lowering activity cannot be expected and has no enlightenment for the present invention. Second, the defatting and dialysis steps are added to the preparation process, that is, the defatting step is added to the pre-treatment of the golden chrysanthemum powder, and the crude protein is dialyzed. The treatment results in an increase in protein content, an improvement in the appearance of crude protein and oligopeptides, and a further increase in lipid-lowering activity. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a golden chrysanthemum oligopeptide and 9-peptide with glycol chains as connecting chains and having lipid-lowering and weight-loss effects, as well as a preparation method and application thereof.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] A golden chrysanthemum oligopeptide with lipid-lowering and weight-loss effects, wherein the oligopeptide has pancreatic lipase inhibitory activity and lipid-lowering activity at the cellular level;

[0011] The inhibition rate of the oligopeptide on pancreatic lipase at concentrations of 2 mg / mL and 0.2 mg / mL is more than 50%;

[0012] The oligopeptide has an inhibitory effect on the accumulation of lipid content in HepG2 cells induced by fatty acids at concentrations of 0.2 mg / mL and 0.02 mg / mL.

[0013] Furthermore, the oligopeptide is obtained by extracting crude protein from crushed golden chrysanthemum petals with or without defatting, and then hydrolyzing it with food-derived hydrolases through dialysis or without dialysis to obtain the protein oligopeptide.

[0014] Furthermore, before extracting protein from petals, the oligopeptide is defatted using an organic solvent such as petroleum ether, n-hexane, ethyl ether, tert-butyl ether, ethanol, methanol, or butanol;

[0015] The protein dialysis condition is a stock solution with a molecular weight cut-off > 3000Da;

[0016] The oligopeptide is obtained by concentrating a stock solution with a molecular weight cutoff of 500 to 5000 Da;

[0017] The food-derived hydrolase is a single enzyme of food-derived protease or a composite enzyme used in combination.

[0018] The preparation method of the golden chrysanthemum oligopeptide as described above comprises the following steps:

[0019] (1) Pretreatment of golden chrysanthemum petals: weighing and crushing the dried golden chrysanthemum petals, sieving, and obtaining golden chrysanthemum crushed material, and extracting protein from the crushed material directly, or extracting protein from the crushed material after defatting with an organic solvent;

[0020] (2) Extraction of crude protein from golden chrysanthemum: the above-mentioned golden chrysanthemum crushed material is mixed with water at a feed ratio of 1:2 to 30 in a g:mL ratio, stirred at a temperature of 0 to 60°C, and a 0.1 to 10 mol / L NaOH aqueous solution is added to adjust the pH to 8.0 to 11.0, and the temperature is maintained and stirred for 1 to 10 hours; cooled to room temperature, centrifuged at 3500 to 8500 rpm for 10 to 20 minutes, the supernatant is taken, the pH is adjusted to 2 to 5 with 0.1 to 1 mol / L HCl, and the liquid is centrifuged at 3500 to 8500 rpm for 10 to 20 minutes, the precipitate is taken, and freeze-dried to obtain peony petal crude protein with a yield of 10 to 20%; the protein is directly enzymatically hydrolyzed by dialysis, or the original solution with a molecular weight cutoff of >3000 Da is dialyzed;

[0021] (3) Preparation of Chrysanthemum chrysanthemum peptide protein: Take the above protein, mix it with water at a feed ratio of g:mL of 1:50-500, add protease for enzymatic hydrolysis, the enzyme activity is 10-1000 KU / g, the enzymatic hydrolysis reaction pH is 3-9, the reaction time is 2-4 h, the reaction temperature is 30-50°C, after the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature for 10-30 min, cool to room temperature, centrifuge at 7000-9000 r / min for 10-30 min, take the supernatant, collect the supernatant below 5 KDa by ultrafiltration, and dry it to obtain Chrysanthemum chrysanthemum petal short peptide protein.

[0022] A 9-peptide contained in the Chrysanthemum chrysanthemum oligopeptide as described above, wherein the sequence of the 9-peptide is AGDTHLGGE, also has pancreatic lipase inhibitory activity; the 9-peptide AGDTHLGGE has a pancreatic lipase inhibition rate of more than 5% at concentrations of 2 mg / mL and 0.2 mg / mL.

[0023] The preparation method of the 9-peptide contained in the golden chrysanthemum oligopeptide as described above comprises the following steps:

[0024] (1) Take 2 g of 2-chlorotrityl resin with a substitution degree of 0.4 mmol / g and swell it with 20 mL of anhydrous dichloromethane. Let it stand for 30 min and transfer the swollen resin into a solid phase synthesis tube;

[0025] (2) 4 mmol of Fmoc-L-Glu(OtBu)-OH and 8 mmol of N,N-diisopropylethylamine were dissolved in 20 mL of anhydrous DCM, stirred thoroughly and poured into a solid phase synthesis tube, which was placed on a shaker for reaction and stirred for 4 h. After the reaction, 10 mmol of chromatographic grade MeOH was added to cap the resin, and the shaker was shaken for 30 min. The resin after the reaction was filtered and washed with 20 mL of dimethylformamide, MeOH, and DCM, respectively.

[0026] (3) Remove the fluorenylmethoxycarbonyl (Fmoc) protecting group from the resin with a mixed solution of 6 mL of morpholine and 14 mL of DMF, twice for 30 min each time, take 10 mg of the resin into a microwave tube, add 1 mL of ninhydrin color developer and 1 mL of phenol, heat to 100 °C, and the reaction is complete when the resin turns blue-purple. Wash the resin with 20 mL of DMF, isopropanol, and DMF, respectively, once for 5 min; the configuration of the ninhydrin color developer is ninhydrin: ethanol in a ratio of 0.2 g:100 mL;

[0027] (4) Dissolve 2.8 mmol of Fmoc-L-Gly and an equivalent amount of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT) and 5.6 mmol of DIPEA in 20 mL of DMF and stir. After the compound is fully dissolved, pour it into a solid phase synthesis tube and place it on a shaker for reaction. Shake for 4 h. Take 10 mg of resin in a microwave tube, add 1 mL of ninhydrin colorimetric agent and 1 mL of phenol, and heat to 100°C. The reaction is complete when the resin turns white. Filter with suction, wash the resin with 20 mL of DMF, MeOH, and DCM, respectively, and store at 4°C.

[0028] Repeat the above peptide grafting reaction in sequence with compounds Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gly-OH, and Fmoc-L-Ala-OH according to the amino acid sequence of the peptide chain. Before each peptide grafting reaction, the Fmoc protecting group at the N-terminus needs to be removed by the deprotection method of step (3);

[0029] (5) Prepare 20 mL of peptide lysis solution, which consists of 5% phenol, 5% ethanedithiol, 2.5% thioanisole, 5% water, and 82.5% trifluoroacetic acid. Mix the five reagents and pour them into a solid phase synthesis tube. Place the tube on a shaker for 4 h of shaking. Filter the lysis solution into 6 times the volume of ice ether and refrigerate overnight. Wash the precipitated peptide with ether 4 times to obtain a white solid peptide AGDTHLGGE. The yield is 65%.

[0030] The preparation was prepared using the Chrysanthemum chrysanthemum petal protein oligopeptide and the 9-peptide AGDTHLGGE as described above.

[0031] Furthermore, the preparation includes powder, granules, powder injection, tablets, capsules or liquid preparations.

[0032] The use of the chrysanthemum petal protein oligopeptide and the 9-peptide AGDTHLGGE as described above in the preparation of foods and / or medicines that provide nutrition for lipid-lowering and weight management.

[0033] The above-mentioned chrysanthemum petal protein oligopeptide and 9-peptide AGDTHLGGE are used in special medical foods, health foods, functional foods and medicines.

[0034] The advantages and positive effects achieved by the present invention are:

[0035] 1. The present invention uses golden chrysanthemum petals as raw materials, obtains plant protein by alkali dissolution and acid precipitation method, and hydrolyzes it by food-derived hydrolase to obtain protein oligopeptides. The plant protein product of the present invention has good safety, and has good pancreatic lipase inhibitory activity and cellular lipid-lowering activity. Therefore, the golden chrysanthemum petal protein oligopeptide provided by the present invention has good effects of lowering blood lipids and controlling body weight, and has application prospects in special medical foods, health foods, functional foods, medicines, and ordinary foods.

[0036] 2. Chrysanthemum has large flowers with yellow color. A large number of broken petals and flowers of poor quality are produced in the process of preparing scented tea. In addition, flowers that are more than two years old will also be treated as waste. The present invention can use Chrysanthemum waste to prepare proteins and oligopeptides, which is a good example of high value-added utilization of agricultural waste and has important research significance and market promotion value.

[0037] 3. The solvent used in the preparation and production process of the golden chrysanthemum oligopeptide of the present invention is water, no organic solvent is discharged, green and environmentally friendly, and suitable for green and environmentally friendly industrial production requirements.

[0038] 4. The present invention provides a green, convenient and higher-protein method for preparing crude protein from Chrysanthemum morifolium and a new application of its enzymatic hydrolysis products.

[0039] 5. The golden chrysanthemum protein and oligopeptide produced by the present invention have uniform color, good fluidity, high yield, simple equipment required for industrial production, and are suitable for industrial production.

[0040] 6. The present invention identified the highest confidence 9-peptide in the golden chrysanthemum oligopeptide by mass spectrometry, and its sequence is AGDTHLGGE, which also has good pancreatic lipase inhibitory activity. The 9-peptide in the present invention has a simple sequence and is easy to prepare, and has the potential to be developed into functional foods and medicines.

[0041] 7. The plant protein oligopeptide produced by the present invention has a small molecular weight and is more easily absorbed and utilized by the human body. While providing nutrition to the human body, it also has a good effect of lowering blood lipids and managing weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The secondary mass spectrum of the oligopeptide AGDTHLGGE identified in the present invention;

[0043] Figure 2 LC-MS mass spectrum of the representative oligopeptide DTIDNVKAK in the present invention. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0045] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0046] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0047] A golden chrysanthemum oligopeptide with lipid-lowering and weight-loss effects, wherein the oligopeptide has pancreatic lipase inhibitory activity and lipid-lowering activity at the cellular level;

[0048] The inhibition rate of the oligopeptide on pancreatic lipase at concentrations of 2 mg / mL and 0.2 mg / mL is more than 50%;

[0049] The oligopeptide has an inhibitory effect on the accumulation of lipid content in HepG2 cells induced by fatty acids at concentrations of 0.2 mg / mL and 0.02 mg / mL.

[0050] Preferably, the oligopeptide is obtained by extracting crude protein from crushed golden chrysanthemum petals with or without defatting, followed by dialysis or without dialysis and then hydrolyzing with food-derived hydrolases to obtain the protein oligopeptide.

[0051] Preferably, before extracting protein from petals, the oligopeptide is defatted using an organic solvent such as petroleum ether, n-hexane, ethyl ether, tert-butyl ether, ethanol, methanol or butanol;

[0052] The protein dialysis condition is a stock solution with a molecular weight cut-off > 3000Da;

[0053] The oligopeptide is obtained by concentrating a stock solution with a molecular weight cutoff of 500 to 5000 Da;

[0054] The food-derived hydrolase is a single enzyme of food-derived protease or a composite enzyme used in combination.

[0055] The preparation method of the golden chrysanthemum oligopeptide as described above comprises the following steps:

[0056] (1) Pretreatment of golden chrysanthemum petals: weighing and crushing the dried golden chrysanthemum petals, sieving, and obtaining golden chrysanthemum crushed material, and extracting protein from the crushed material directly, or extracting protein from the crushed material after defatting with an organic solvent;

[0057] (2) Extraction of crude protein from golden chrysanthemum: the above-mentioned golden chrysanthemum crushed material is mixed with water at a feed ratio of 1:2 to 30 in a g:mL ratio, stirred at a temperature of 0 to 60°C, and a 0.1 to 10 mol / L NaOH aqueous solution is added to adjust the pH to 8.0 to 11.0, and the temperature is maintained and stirred for 1 to 10 hours; cooled to room temperature, centrifuged at 3500 to 8500 rpm for 10 to 20 minutes, the supernatant is taken, the pH is adjusted to 2 to 5 with 0.1 to 1 mol / L HCl, and the liquid is centrifuged at 3500 to 8500 rpm for 10 to 20 minutes, the precipitate is taken, and freeze-dried to obtain peony petal crude protein with a yield of 10 to 20%; the protein is directly enzymatically hydrolyzed by dialysis, or the original solution with a molecular weight cutoff of >3000 Da is dialyzed;

[0058] (3) Preparation of Chrysanthemum chrysanthemum peptide protein: Take the above protein, mix it with water at a feed ratio of g:mL of 1:50-500, add protease for enzymatic hydrolysis, the enzyme activity is 10-1000 KU / g, the enzymatic hydrolysis reaction pH is 3-9, the reaction time is 2-4 h, the reaction temperature is 30-50°C, after the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature for 10-30 min, cool to room temperature, centrifuge at 7000-9000 r / min for 10-30 min, take the supernatant, collect the supernatant below 5 KDa by ultrafiltration, and dry it to obtain Chrysanthemum chrysanthemum petal short peptide protein.

[0059] A 9-peptide contained in the Chrysanthemum chrysanthemum oligopeptide as described above, wherein the sequence of the 9-peptide is AGDTHLGGE, also has pancreatic lipase inhibitory activity; the 9-peptide AGDTHLGGE has a pancreatic lipase inhibition rate of more than 5% at concentrations of 2 mg / mL and 0.2 mg / mL.

[0060] The preparation method of the 9-peptide contained in the golden chrysanthemum oligopeptide as described above comprises the following steps:

[0061] (1) Take 2 g of 2-chlorotrityl resin with a substitution degree of 0.4 mmol / g and swell it with 20 mL of anhydrous dichloromethane. Let it stand for 30 min and transfer the swollen resin into a solid phase synthesis tube;

[0062] (2) 4 mmol of Fmoc-L-Glu(OtBu)-OH and 8 mmol of N,N-diisopropylethylamine were dissolved in 20 mL of anhydrous DCM, stirred thoroughly and poured into a solid phase synthesis tube, which was placed on a shaker for reaction and stirred for 4 h. After the reaction, 10 mmol of chromatographic grade MeOH was added to cap the resin, and the shaker was shaken for 30 min. The resin after the reaction was filtered and washed with 20 mL of dimethylformamide, MeOH, and DCM, respectively.

[0063] (3) Remove the fluorenylmethoxycarbonyl (Fmoc) protecting group from the resin with a mixed solution of 6 mL of morpholine and 14 mL of DMF, twice for 30 min each time, take 10 mg of the resin into a microwave tube, add 1 mL of ninhydrin color developer and 1 mL of phenol, heat to 100 °C, and the reaction is complete when the resin turns blue-purple. Wash the resin with 20 mL of DMF, isopropanol, and DMF, respectively, once for 5 min; the configuration of the ninhydrin color developer is ninhydrin: ethanol in a ratio of 0.2 g:100 mL;

[0064] (4) Dissolve 2.8 mmol of Fmoc-L-Gly and an equivalent amount of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT) and 5.6 mmol of DIPEA in 20 mL of DMF and stir. After the compound is fully dissolved, pour it into a solid phase synthesis tube and place it on a shaker for reaction. Shake for 4 h. Take 10 mg of resin in a microwave tube, add 1 mL of ninhydrin colorimetric agent and 1 mL of phenol, and heat to 100°C. The reaction is complete when the resin turns white. Filter with suction, wash the resin with 20 mL of DMF, MeOH, and DCM, respectively, and store at 4°C.

[0065] Repeat the above peptide grafting reaction in sequence with compounds Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gly-OH, and Fmoc-L-Ala-OH according to the amino acid sequence of the peptide chain. Before each peptide grafting reaction, the Fmoc protecting group at the N-terminus needs to be removed by the deprotection method of step (3);

[0066] (5) Prepare 20 mL of peptide lysis solution, which consists of 5% phenol, 5% ethanedithiol, 2.5% thioanisole, 5% water, and 82.5% trifluoroacetic acid. Mix the five reagents and pour them into a solid phase synthesis tube. Place the tube on a shaker for 4 h of shaking. Filter the lysis solution into 6 times the volume of ice ether and refrigerate overnight. Wash the precipitated peptide with ether 4 times to obtain a white solid peptide AGDTHLGGE. The yield is 65%.

[0067] The preparation was prepared using the Chrysanthemum chrysanthemum petal protein oligopeptide and the 9-peptide AGDTHLGGE as described above.

[0068] Preferably, the preparation includes powder, granules, powder injection, tablet, capsule or liquid preparation.

[0069] The use of the chrysanthemum petal protein oligopeptide and the 9-peptide AGDTHLGGE as described above in the preparation of foods and / or medicines that provide nutrition for lipid-lowering and weight management.

[0070] The above-mentioned chrysanthemum petal protein oligopeptide and 9-peptide AGDTHLGGE are used in special medical foods, health foods, functional foods and medicines.

[0071] Specifically, the relevant preparation and detection are as follows:

[0072] Example 1 A golden chrysanthemum oligopeptide, the preparation steps of which are as follows:

[0073] S1: The dried golden chrysanthemum flowers were crushed and sieved (60-100 meshes), 10g of golden chrysanthemum crushed material was mixed with 300mL of water, heated and stirred at 55°C for 30min, and pH was adjusted to 8.5 with 1mol / L NaOH aqueous solution, and the temperature was maintained and stirred for 5h. After cooling to room temperature, centrifuged at 8000rpm for 12min, the supernatant was taken, and the pH of the supernatant was adjusted to 4.0 with 1mol / L dilute hydrochloric acid, and the liquid was centrifuged at 8000rpm for 12min, the precipitate was taken, and vacuum freeze-dried to obtain golden chrysanthemum crude protein. 500mg of crude protein was added to 50mL of water, and pH was adjusted to 4.0 with 1mol / L dilute hydrochloric acid, and then centrifuged at 8000rpm for 12min, the precipitate was taken, and vacuum freeze-dried, and the yield was about 10%.

[0074] S2: Take 400 mg of the above-mentioned golden chrysanthemum protein, add 60 mL of water, stir at 55°C for 10 min, adjust pH to 8.0 with 1 mol / L NaOH aqueous solution, add 100 mg of flavor protease (enzyme activity unit 40 KU / g), enzymolysis for 2.5 h, heat to 90°C to inactivate the enzyme for 20 min after the enzymolysis, cool to 55°C, adjust pH to 8.0 with 1 mol / L NaOH aqueous solution, add 10 mg of papain (enzyme activity unit 800 KU / g), enzymolysis again for 2 h, heat to 90°C to inactivate the enzyme for 20 min after the enzymolysis, cool to room temperature, centrifuge at 8000 rpm for 12 min, take the supernatant, collect the supernatant below 3 KDa by ultrafiltration, and vacuum freeze-dry to obtain the composite enzymatically hydrolyzed golden chrysanthemum oligopeptide.

[0075] Example 2 A golden chrysanthemum oligopeptide, the preparation steps of which are as follows:

[0076] S1: The dried golden chrysanthemum flowers were crushed and sieved (60-100 meshes), 20g of golden chrysanthemum crushed material was taken, petroleum ether was added for degreasing, the solid-liquid ratio was 1:10 (g:mL), stirred at room temperature for 1h, filtered to obtain the filter cake, the filter cake was mixed with petroleum ether again, stirred at room temperature for 1h, filtered to obtain the filter cake, and the filter cake was dried to obtain 19g of defatted golden chrysanthemum crushed material. 10g of defatted golden chrysanthemum crushed material was mixed with 300mL of water, heated and stirred at 55℃ for 30min, adjusted pH to 8.5 with 1mol / L NaOH aqueous solution, and continued to stir at the temperature for 5h. After cooling to room temperature, centrifuged at 8000rpm for 12min, the supernatant was taken, the supernatant was adjusted to pH = 4.0 with 1mol / L dilute hydrochloric acid, and the liquid was centrifuged at 8000rpm for 12min, the precipitate was taken, and vacuum freeze-dried to obtain golden chrysanthemum crude protein. Take 500 mg of crude protein and put it into a 3Kda dialysis bag, add 50 mL of water and dialyze for 12 hours, adjust the dialyzed liquid to pH 4.0 with 1 mol / L dilute hydrochloric acid, then centrifuge at 8000 rpm for 12 minutes, take the precipitate, and freeze-dry it in vacuum, with a yield of about 6%. Although the yield of crude protein extracted from golden chrysanthemum by petroleum ether degreasing is reduced, the appearance of the protein is significantly improved, and it is a light gray fluid powder.

[0077] S2: Take 400 mg of the above-mentioned golden chrysanthemum protein, add 60 mL of water, stir at 55°C for 10 min, adjust pH to 8.0 with 1 mol / L NaOH aqueous solution, add 100 mg of flavor protease (enzyme activity unit 40 KU / g), enzymolysis for 2.5 h, heat to 90°C to inactivate the enzyme for 20 min after the enzymolysis, cool to 55°C, adjust pH to 8.0 with 1 mol / L NaOH aqueous solution, add 10 mg of papain (enzyme activity unit 800 KU / g), enzymolysis again for 2 h, heat to 90°C to inactivate the enzyme for 20 min after the enzymolysis, cool to room temperature, centrifuge at 8000 rpm for 12 min, take the supernatant, collect the supernatant below 3 KDa by ultrafiltration, and vacuum freeze-dry to obtain the composite enzymatically hydrolyzed golden chrysanthemum oligopeptide.

[0078] Example 3 A golden chrysanthemum oligopeptide, the preparation steps of which are as follows:

[0079] The preparation process of golden chrysanthemum crude protein and the hydrolysis process of oligopeptides are the same as those in Example 1, except that the defatting solvent is changed from petroleum ether to ethanol. The yield of crude protein is 7%, and the appearance color and fluidity of crude protein are better than those of crude protein that has not been defatted.

[0080] Example 4 A golden chrysanthemum oligopeptide, the preparation steps of which are as follows:

[0081] The preparation process of golden chrysanthemum crude protein and the hydrolysis process of oligopeptides are the same as those in Example 1, except that the defatting solvent is changed from petroleum ether to n-hexane. The yield of crude protein is 8%, and the appearance color and fluidity of crude protein are better than those of crude protein that has not been defatted.

[0082] Example 5 Pancreatic lipase inhibition experiment of golden chrysanthemum protein and oligopeptide

[0083] Dissolve tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) in deionized water, gradually adjust the pH to 8.0 with dilute hydrochloric acid, and dilute to 200 mL to prepare a 13 mM buffer. Pancreatic lipase is dissolved in the above Tris-HCl buffer to form a 1 mg / mL working solution. Weigh the test compound and the positive control orlistat, dissolve in dimethyl sulfoxide (DMSO), the final concentration of the test compound is 2 mg / mL and 0.2 mg / mL, and the final concentration of the positive control orlistat is 0.08 μM. To prepare the stop solution, weigh citric acid and sodium citrate powders, respectively, and prepare a 0.1 mol / L solution with distilled water, slowly add citric acid solution to the sodium citrate solution, and adjust pH = 4.2. The substrate 4-methylumbelliferyl oleate (4-MUO) is dissolved in DMSO, diluted to a concentration of 0.1 mM, and stored in the dark for use.

[0084] The test system is as follows: blank background group (A1): Tris-HCl buffer (50 μL) + substrate (50 μL) + sodium citrate stop solution (100 μL); blank group (A2): Tris-HCl buffer (25 μL) + substrate (50 μL) + pancreatic lipase reaction solution (25 μL) + sodium citrate stop solution (100 μL); compound background group (A3): Tris-HCl buffer (40 μL) + substrate (50 μL) + compound (10 μL) + sodium citrate stop solution (100 μL); compound test group (A4): Tris-HCl buffer (15 μL) + substrate (50 μL) + compound (10 μL) + pancreatic lipase reaction solution (25 μL) + sodium citrate stop solution (100 μL).

[0085] The test method is as follows: add the above-mentioned solutions to a 96-well plate in order to mix the liquids evenly. After adding the enzyme solution, react at 25°C for 30 minutes, add 100 μL of sodium citrate reaction termination solution to each well, and measure the OD value at an excitation wavelength of 360±20 nm and an emission wavelength of 460±20 nm. The calculation formula for the pancreatic lipase inhibition rate of the compound is as follows: Pancreatic lipase inhibition rate % = ((A2-A1)-(A4-A3)) / (A2-A1)*100.

[0086] The results of pancreatic lipase inhibitory activity are shown in Table 1.

[0087] Table 1 Pancreatic lipase activity test results

[0088]

[0089] Note: a All tests are the average of three independent experiments and two replicates. b Orlistat is a pancreatic lipase inhibitor used clinically, with a test concentration of 0.08 μM.

[0090] As shown in Table 1, the crude protein of golden chrysanthemum extracted by petroleum ether and n-hexane defatting not only has a significantly improved appearance, but also has a high pancreatic lipase inhibitory activity after compound enzymatic hydrolysis. The activity of the oligopeptide defatted by n-hexane is the highest, especially at a concentration of 0.2 mg / mL, the inhibition rate is higher than 80%. Pancreatic lipase inhibitors play an important role in blood lipid control and weight control. Therefore, this type of substance has a good application prospect in the field of lowering blood lipids and losing weight.

[0091] Example 6 Determination of protein content of Chrysanthemum chinense oligopeptide

[0092] Since n-hexane defatting significantly improves lipid-lowering activity, the protein content of the samples was tested. The protein content was determined by Kjeldahl nitrogen determination method in accordance with GB 5009.5-2016. The results are shown in Table 2.

[0093] Table 2 Comparison of protein content

[0094] serial number Protein extraction conditions Egg content (%) Example 1 No delipidation, no dialysis 19.23 Example 4 Hexane degreasing + dialysis 56.36

[0095] As shown in Table 2, the chrysanthemum short peptide protein content in Example 1 without defatting and dialysis is 19.23%, and after defatting and dialysis, the chrysanthemum short peptide protein content in Example 4 is 56.36%. The results show that the protein content is significantly improved after defatting and dialysis. In addition, the appearance color of the protein and oligopeptide obtained in Example 4 is more uniform than that in Example 1, and the fluidity is better.

[0096] Example 7 Determination of lipid-lowering activity of Chrysanthemum chrysantha oligopeptide at cellular level

[0097] Weigh sodium oleate and sodium palmitate powders, dissolve them with 0.1M sodium hydroxide solution, and prepare solutions with a concentration of 200mM. Add 1% bovine serum albumin (BSA) to DMEM low-glucose medium (containing 1g / L glucose) until dissolved. Mix the above-prepared DMEM low-glucose culture medium, sodium oleate solution and sodium palmitate solution in a volume ratio of 400:2:1, filter and sterilize with a 0.45μm filter membrane to obtain long-chain fatty acid inducers (FFAs). Prepare another tube of DMEM low-glucose medium containing only 1% BSA as a blank control.

[0098] Weigh Oil Red O powder and dissolve it in isopropanol to make a 10 mg / mL stock solution. Mix the Oil Red O stock solution and water in a volume ratio of 3:2, filter and sterilize with a 0.45 μm filter membrane to obtain Oil Red O working solution. Dissolve the paraformaldehyde powder in 1× PBS to obtain a 4% paraformaldehyde solution.

[0099] Method for determining cell lipid content: HepG2 cells were cultured at 1×10 5 The cells were inoculated in a 6-well plate at a density of 2 mL per well. After culturing for 24 hours, the cells were washed three times with 1×PBS and replaced with DMEM serum-free high-glucose medium containing 4.5 g / L glucose. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for another 24 hours. After washing three times with 1×PBS, the cells were replaced with FFAs inducers to stimulate intracellular fat accumulation. The blank control group was added with DMEM low-glucose medium containing 1% BSA, and different concentrations of compound solutions (10 μL) were added according to the system volume and compound solution volume of 200:1. The final concentration of the compound was 0.2 mg / mL and 0.02 mg / mL. The negative control group was added with an equal volume of solvent (10 μL). After 24 hours, the original liquid was aspirated and washed three times with 1×PBS. 2 mL of 4% paraformaldehyde solution was added to each well to fix the cells for 30 minutes. The cells were washed three times with 1×PBS and 2 mL of isopropanol solution was added to each well for 5 minutes to fix the cells. The isopropanol was aspirated and 2 mL of Oil Red O working solution was added to each well to stain for 1 hour in the dark. The cells were washed four times with distilled water, 1 mL of isopropanol solution was added to each well, and the cells were washed out by shaking at 37°C for 10 min. The changes in intracellular lipid content were reflected by detecting the absorbance at a wavelength of 492 nm.

[0100] The calculation formula of intracellular lipid content is as follows:

[0101] Fat content% = (A 化合物组 -A 空白对照组 ) / (A 阴性对照组 -A 空白对照组 )*100

[0102] The effect of chrysanthemum oligopeptides on the lipid content of HepG2 cells induced by fatty acids, due to solubility limitations, the upper limit concentration is 0.2 mg / mL, as shown in Table 3.

[0103] Table 3 Effects of chrysanthemum oligopeptides on lipid content in HepG2 cells

[0104]

[0105] Note: a All tests are the average of three independent experiments and two replicates. b Lovastatin is a clinically used lipid-lowering drug, and the test concentration is 10 μM.

[0106] It can be seen from Table 3 that at a concentration of 0.2 mg / mL, the chrysanthemum oligopeptides of Examples 1 and 4 both have certain lipid-lowering activity, and after n-hexane defatting and dialysis, the oligopeptide in Example 4 also has obvious lipid-lowering activity at a concentration of 0.02 mg / mL. The test concentration of lovastatin is 10 μM, and its activity is equivalent to that of Example 4 at a concentration of 0.2 mg / mL.

[0107] Example 8 Identification of polypeptide sequences of chrysanthemum enzymatic protein

[0108] The short peptide protein of Chrysanthemum chrysanthemum obtained by enzymatic hydrolysis in Example 4 was used for polypeptide sequence identification. The experiment was determined using Thermo EASYnLC liquid chromatograph and Thermo Scientific QE HF mass spectrometer. The polypeptide was fully dissolved with 0.1% trifluoroacetic acid (TFA) aqueous solution, and the microchromatography column (TIP) was rinsed 10 times with 50μL of 60% acetonitrile (ACN) / 0.1% TFA. Then, the TIP was washed 10 times with 10μL of 0.1% TFA. The sample was aspirated and discharged from the TIP 20 times, and the liquid was discharged. The TIP was washed 5 times with 10μL of 0.1% TFA. The peptide segment was eluted into a new EP tube with 10μL of 60% ACN and 0.1% TFA, and vacuum dried and stored after the desalting process was completed. The desalted peptide was dissolved in 20 μL of dissolving solution (0.1% formic acid, 5% acetonitrile), vortexed thoroughly, centrifuged at 13500 rpm and 4°C for 20 min, the supernatant was transferred to a sample tube, and 8 uL was aspirated for mass spectrometry identification; the mobile phase included (A) 0.1% formic acid aqueous solution and (B) 0.1% formic acid 80% acetonitrile solution.

[0109] The LC linear gradient was as follows: 3% B for 3 min, 3% B to 8% B for 4 min, 8% B to 32% B for 3 min, 32% B to 44% B for 5 min, 44% B to 99% B for 5 min, 99% B for 4 min, and finally flushing the line for 5 min at 3% B. The parent ion range was set to 350–1550 m / z.

[0110] Peaks Studio software was used to analyze and search the raw mass spectrometry (MS) data to identify protein modifications and enzyme cleavage sites. By querying the Uniprot database and comparing it with the protein database (UNIPROT_taxonomy_id13422.fasta), we focused on major protein modifications such as acetylation (variable at the N-terminus), oxidation (variable), and carbamidomethylation (fixed at the C-terminus). The primary mass spectrometry error was set to 20 ppm to accommodate the mass deviation of the instrument measurement; the secondary mass spectrometry error was set to 0.05 Da to accurately match the mass of the fragment ions.

[0111] The peptide results with the highest confidence in the sequence identification results are as follows:

[0112] Table 4 Mass spectrometry identification results of Chrysanthemum chrysantha

[0113] Peptide sequence -10lgP Molecular weight length Mass-to-nuclear ratio (m / z) Retention time (min) AGDTHLGGE 47.35 855.3723 9 428.6948 12.47

[0114] As shown in Table 4, the peptide sequence with the highest confidence in mass spectrometry identification is AGDTHLGGE, and the corresponding identification result is shown in the mass spectrum ( Figure 1 ), molecular weight is 855.3723, retention time is 12.47min.

[0115] Example 9 Identification of Solid Phase Synthesis of Peptide AGDTHLGGE

[0116] In order to further test the activity of the polypeptide sequence, the present invention synthesized the polypeptide using solid phase synthesis. The steps are as follows:

[0117] S1: Take 2 g of 2-chlorotrityl resin (substitution degree 0.4 mmol / g) and swell it with 20 mL of anhydrous dichloromethane (DCM), let it stand for 30 minutes, and after swelling, transfer the resin into a solid phase synthesis tube.

[0118] S2: Dissolve 4mmol of Fmoc-L-Glu(OtBu)-OH and 8mmol of N,N-diisopropylethylamine (DIPEA) in 20mL of anhydrous DCM, stir thoroughly and pour into a solid phase synthesis tube, place the solid phase synthesis tube on a shaker for reaction, and stir for 4h. After the reaction, add 10mmol of chromatographic grade MeOH to cap the resin, and shake on a shaker for 30min. Filter the resin after the reaction, and wash the resin once with 20mL of dimethylformamide (DMF), MeOH, and DCM in turn.

[0119] S3: Remove the fluorenylmethoxycarbonyl (Fmoc) protecting group from the resin with a mixed solution of 6 mL of morpholine and 14 mL of DMF, twice for 30 min each time, put 10 mg of the resin in a microwave tube, add 1 mL of ninhydrin color developer and 1 mL of phenol, heat to 100°C, and the reaction is complete when the resin turns blue-purple (the configuration of ninhydrin color developer is ninhydrin: ethanol = 0.2 g: 100 mL), wash the resin with 20 mL of DMF, isopropanol, and DMF once each, each for 5 min.

[0120] S4: Dissolve 2.8 mmol of Fmoc-L-Gly and an equivalent amount of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT) and 5.6 mmol of DIPEA in 20 mL of DMF and stir. After the compound is fully dissolved, pour it into a solid phase synthesis tube and place it in a shaker for reaction. Shake for 4 hours. Take 10 mg of resin in a microwave tube, add 1 mL of ninhydrin color developer and 1 mL of phenol, heat to 100°C, and the reaction is complete when the resin turns white. Filter it and wash the resin once with 20 mL of DMF, MeOH and DCM in turn, and store it at 4°C.

[0121] According to the amino acid sequence of the peptide chain, the above peptide grafting reaction was repeated with compounds Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gly-OH, and Fmoc-L-Ala-OH in sequence. Before each peptide grafting reaction, the Fmoc protecting group at the N-terminus was removed by the deprotection method in step 3.

[0122] After Fmoc-L-Ala-OH was connected to the resin and the Fmoc protecting group was removed, the resin was washed with 20 mL of DMF, MeOH, and DCM, respectively, for 5 min each, and then washed with 20 mL of DCM for 3 times, for 5 min each.

[0123] S5: Prepare 20 ml of polypeptide lysate, which consists of phenol (5%), ethanedithiol (5%), thioanisole (2.5%), water (5%), and trifluoroacetic acid (82.5%). Mix the five reagents and pour them into a solid phase synthesis tube. Place them on a shaker for 4 hours of shaking. Filter the lysate into ice ether 6 times the volume of the lysate and refrigerate overnight. Centrifuge the precipitated polypeptide at 4°C, control the speed to 8000rpm, discard the supernatant, add a small amount of ether or methyl tert-butyl ether and centrifuge again after ultrasonic shaking. Repeat the operation to wash the polypeptide for more than 6 times. After the remaining polypeptide is evaporated and removed by ether, add a small amount of water to dissolve it, freeze-dry it, and obtain a white solid polypeptide AGDTHLGGE. The yield is about 65%.

[0124] The above peptides were verified by LS-MS, HPLC conditions: flow rate 0.2mL / min, injection volume: 1μL, column oven 25℃. Mobile phase A is water, phase B is methanol (HPLC grade). Isocratic elution: 0-6min, 100% B. Mass spectrometry conditions: In the range of m / z 50-1700, mass spectra were obtained simultaneously by electrospray ionization (ESI) at low and high fragmentation voltages (-3.5KV and 4.5KV). The mass spectrometry results are shown in Figure 2The molecular ion peak is 894.3357, which is [M+K] + The positive ion peak is consistent with the molecular weight of 855.3723 identified in Table 4.

[0125] The peptide was further tested for lipid-lowering activity, and the results are as follows:

[0126] Table 5. Hypolipidemic activity of peptide AGDTHLGGE

[0127]

[0128] Note: a All tests are the average of three independent experiments and two replicates. b Orlistat is a pancreatic lipase inhibitor used clinically, with a test concentration of 0.08 μM.

[0129] As shown in Table 5, the peptide with the highest confidence level also has good pancreatic lipase inhibitory activity, which is basically equivalent to the activity of the peptide mixture with the same concentration. The peptide sequence is simple, easy to synthesize and industrialize, and has good application prospects.

[0130] In summary, there is no report on the lipid-lowering activity of golden chrysanthemum oligopeptides in the prior art, and the present invention is the first to discover that such substances have lipid-lowering activity. In addition, there are no defatting and dialysis processing steps in the preparation process of golden chrysanthemum oligopeptides in the prior art. The present invention adds a defatting step to the pre-treatment of golden chrysanthemum powder, and dialyzes the crude protein. The treatment results in an increase in egg content, and the appearance and fluidity are also improved, and the lipid-lowering activity is further improved. In addition, golden chrysanthemum in the present invention has a long history of consumption, and the hydrolases used in the proteins and oligopeptides prepared therefrom are all food-derived enzymes. The safety of proteins and oligopeptides is high, and compared with lipid-lowering drugs, they are more suitable for long-term consumption. Therefore, golden chrysanthemum protein and oligopeptides can be used as lipid-lowering and weight-loss raw materials, and can be used for the addition of medicines, health foods, special medical foods and foods, and have good practical application value and market potential.

[0131] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A golden chrysanthemum oligopeptide with the effects of lowering blood lipids and losing weight, characterized in that: The oligopeptide has pancreatic lipase inhibitory activity and cellular level lipid-lowering activity; The inhibition rate of the oligopeptide on pancreatic lipase at concentrations of 2 mg / mL and 0.2 mg / mL is more than 50%; The oligopeptide has an inhibitory effect on the accumulation of lipid content in HepG2 cells induced by fatty acids at concentrations of 0.2 mg / mL and 0.02 mg / mL.

2. The golden chrysanthemum oligopeptide according to claim 1, characterized in that: The oligopeptide is obtained by extracting crude protein from crushed golden chrysanthemum petals after defatting or not, and then hydrolyzing it with food-derived hydrolases after dialysis or not.

3. The golden chrysanthemum oligopeptide according to claim 2, characterized in that: Before the oligopeptide is extracted from the petals for protein extraction, the solvent used for defatting is an organic solvent such as petroleum ether, n-hexane, ethyl ether, tert-butyl ether, ethanol, methanol, or butanol; The protein dialysis condition is a stock solution with a molecular weight cut-off > 3000Da; The oligopeptide is obtained by concentrating a stock solution with a molecular weight cutoff of 500 to 5000 Da; The food-derived hydrolase is a single enzyme of food-derived protease or a composite enzyme used in combination.

4. The method for preparing the golden chrysanthemum oligopeptide according to any one of claims 1 to 3, characterized in that: The steps include: (1) Pretreatment of golden chrysanthemum petals: weighing and crushing the dried golden chrysanthemum petals, sieving, and obtaining golden chrysanthemum crushed material, and extracting protein from the crushed material directly, or extracting protein from the crushed material after defatting with an organic solvent; (2) Extraction of crude protein from golden chrysanthemum: the above-mentioned golden chrysanthemum crushed material is mixed with water at a feed ratio of 1:2 to 30 in a g:mL ratio, stirred at a temperature of 0 to 60°C, and a 0.1 to 10 mol / L NaOH aqueous solution is added to adjust the pH to 8.0 to 11.0, and the temperature is maintained and stirred for 1 to 10 hours; cooled to room temperature, centrifuged at 3500 to 8500 rpm for 10 to 20 minutes, the supernatant is taken, the pH is adjusted to 2 to 5 with 0.1 to 1 mol / L HCl, and the liquid is centrifuged at 3500 to 8500 rpm for 10 to 20 minutes, the precipitate is taken, and freeze-dried to obtain peony petal crude protein with a yield of 10 to 20%; the protein is directly enzymatically hydrolyzed by dialysis, or the original solution with a molecular weight cutoff of >3000 Da is dialyzed; (3) Preparation of Chrysanthemum chrysanthemum peptide protein: Take the above protein, mix it with water at a feed ratio of g:mL of 1:50-500, add protease for enzymatic hydrolysis, the enzyme activity is 10-1000 KU / g, the enzymatic hydrolysis reaction pH is 3-9, the reaction time is 2-4 h, the reaction temperature is 30-50°C, after the enzymatic hydrolysis is completed, inactivate the enzyme at high temperature for 10-30 min, cool to room temperature, centrifuge at 7000-9000 r / min for 10-30 min, take the supernatant, collect the supernatant below 5 KDa by ultrafiltration, and dry it to obtain Chrysanthemum chrysanthemum petal short peptide protein.

5. A nonapeptide contained in the golden chrysanthemum oligopeptide according to any one of claims 1 to 4, characterized in that: The sequence of the 9-peptide is AGDTHLGGE, which also has pancreatic lipase inhibitory activity; the inhibition rate of the 9-peptide AGDTHLGGE on pancreatic lipase is more than 5% at concentrations of 2 mg / mL and 0.2 mg / mL.

6. The method for preparing the 9-peptide contained in the golden chrysanthemum oligopeptide as claimed in claim 5, characterized in that: The preparation steps are as follows: (1) Take 2 g of 2-chlorotrityl resin with a substitution degree of 0.4 mmol / g and swell it with 20 mL of anhydrous dichloromethane. Let it stand for 30 min and transfer the swollen resin into a solid phase synthesis tube; (2) 4 mmol of Fmoc-L-Glu(OtBu)-OH and 8 mmol of N,N-diisopropylethylamine were dissolved in 20 mL of anhydrous DCM, stirred thoroughly and poured into a solid phase synthesis tube, which was placed on a shaker for reaction and stirred for 4 h. After the reaction, 10 mmol of chromatographic grade MeOH was added to cap the resin, and the shaker was shaken for 30 min. The resin after the reaction was filtered and washed with 20 mL of dimethylformamide, MeOH, and DCM, respectively. (3) Remove the fluorenylmethoxycarbonyl protecting group from the resin with a mixed solution of 6 mL of morpholine and 14 mL of DMF, twice for a total of 30 min each time, take 10 mg of the resin into a microwave tube, add 1 mL of ninhydrin color developer and 1 mL of phenol, heat to 100 ° C, and the reaction is complete when the resin turns blue-purple. Wash the resin with 20 mL of DMF, isopropanol, and DMF, respectively, once for 5 min; wherein, the configuration of the ninhydrin color developer is ninhydrin: ethanol ratio of 0.2 g: 100 mL; (4) Dissolve 2.8 mmol of Fmoc-L-Gly and an equivalent amount of O-benzotriazole-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and 5.6 mmol of DIPEA in 20 mL of DMF and stir. After the compound is fully dissolved, pour it into a solid phase synthesis tube and place it on a shaker for reaction. Shake for 4 h. Take 10 mg of resin in a microwave tube, add 1 mL of ninhydrin colorimetric agent and 1 mL of phenol, and heat to 100°C. The reaction is complete when the resin turns white. Filter it with suction, wash the resin with 20 mL of DMF, MeOH, and DCM, respectively, and store it at 4°C. Repeat the above peptide grafting reaction in sequence with compounds Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gly-OH, and Fmoc-L-Ala-OH according to the amino acid sequence of the peptide chain. Before each peptide grafting reaction, the Fmoc protecting group at the N-terminus needs to be removed by the deprotection method of step (3); (5) Prepare 20 mL of peptide lysis solution, which consists of 5% phenol, 5% ethanedithiol, 2.5% thioanisole, 5% water, and 82.5% trifluoroacetic acid. Mix the five reagents and pour them into a solid phase synthesis tube. Place the tube on a shaker for 4 h of shaking. Filter the lysis solution into 6 times the volume of ice ether and refrigerate overnight. Wash the precipitated peptide with ether 4 times to obtain a white solid peptide AGDTHLGGE. The yield is 65%.

7. A preparation prepared using the Chrysanthemum chrysanthemum petal protein oligopeptide and the 9-peptide AGDTHLGGE as described in any one of claims 1 to 4.

8. The preparation according to claim 7, characterized in that: The preparations include powders, granules, powder injections, tablets, capsules or liquid preparations.

9. Use of the golden chrysanthemum petal protein oligopeptide and the nonapeptide AGDTHLGGE as claimed in any one of claims 1 to 4 in the preparation of foods and / or medicines that provide nutrition for lipid-lowering and weight management.

10. Use of the golden chrysanthemum petal protein oligopeptide and the nonapeptide AGDTHLGGE as claimed in any one of claims 1 to 4 in special medical foods, health foods, functional foods and medicines.

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