An intestinal absorbable cholesterol-lowering peptide and its solid-phase synthesis method and application

Abstract

The present invention discloses an intestinal absorbable cholesterol-lowering peptide, a solid-phase synthesis method, and its application. The cholesterol-lowering peptide has an amino acid sequence of His-Thr-Ser-Gly-Tyr (HTSGY), which is derived from whey protein and is an intestinal absorbable pentapeptide. The peptide can significantly reduce cholesterol levels in the human body, effectively alleviate atherosclerosis, and has the potential to reduce the incidence of hepatobiliary diseases. The peptide is of great significance for the development of dairy products, animal feed, health products, health foods, and medicines with cholesterol-lowering functions, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of active peptides, and in particular relates to an intestinal absorbable cholesterol-lowering peptide and a solid-phase synthesis method and application thereof. Background Art

[0002] Modern medical research has demonstrated that unhealthy lifestyles and unbalanced nutrition contribute to excessive cholesterol intake. High rates of coronary heart disease, hyperlipidemia, and arteriosclerosis are linked to elevated cholesterol levels. Current treatment in my country primarily relies on targeted drug therapies, but these are expensive and often come with significant side effects. Ideally, dietary supplementation with exogenous cholesterol-lowering agents is the best way to maintain health and protect the body from the harmful effects of cholesterol. Enzymatic hydrolysis of certain proteins yields small peptides with cholesterol-lowering activity. These peptides can be developed into cholesterol-lowering functional foods or as alternatives to synthetic drugs. Natural cholesterol-lowering substances offer advantages over synthetic cholesterol-lowering agents and have become a hot topic of research for food safety.

[0003] Whey protein is a general term for a certain type of protein. It is the highest content component in the whey discharged after being processed into cheese. It mainly includes β -Lactoglobulin ( β -lactoglobulin), α - Lactalbumin ( α Whey protein, known as the "king of proteins," is not only high in protein, low in fat and cholesterol, and highly digestible, but also contains 20 essential fatty acids and boasts a comprehensive and optimal amino acid profile. Whey protein also contains numerous functional ingredients, such as lactoferrin, glycomacropeptides, lactoperoxidase, and growth factors. Protease hydrolysis of whey protein can produce whey protein hydrolysates with specific biological properties. Studies have shown that whey protein hydrolysates exhibit ACE inhibitory activity, immune activity, anti-inflammatory, hypoglycemic, and antioxidant properties, some of which are already in industrial production.

[0004] Caco-2 cells are colon and rectal cancer cells derived from humans. Under appropriate culture conditions, they can differentiate into a monolayer of small intestinal epithelial cells on a polycarbonate membrane. These cells possess properties very similar to those of small intestinal cells, such as cell polarity and tight junctions, a microvilli structure, and the presence of certain active transport systems for carbohydrates, amino acids, and dipeptides. Due to their close similarity in morphology and properties to small intestinal epithelial cells, the Caco-2 cell model is a commonly used in vitro absorption model, effectively serving as a foundation for studying the absorption of drugs, peptides, and other substances. Furthermore, intestinal transport models constructed using Caco-2 cells are often used to screen for peptides that can be fully digested and absorbed by the intestine. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides an intestinal absorbable cholesterol-lowering peptide and its solid-phase synthesis method and application. The cholesterol-lowering peptide has an amino acid sequence of His-Thr-Ser-Gly-Tyr, can be absorbed by the intestine, and can be used to prepare drugs or health products for lowering cholesterol content, and has good market prospects.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A cholesterol-lowering peptide (HTSGY) that can be absorbed by the intestine has an amino acid sequence of His-Thr-Ser-Gly-Tyr. The cholesterol-lowering peptide can be absorbed by the intestine.

[0008] The solid-phase synthesis method of the intestinal absorbable cholesterol-lowering peptide comprises swelling and washing a dichlororesin, removing the Fmoc protecting group, adding an amino acid for a condensation reaction, and repeating the removal-protection-condensation process until all amino acids are connected; cutting the resin to obtain a crude His-Thr-Ser-Gly-Tyr polypeptide, and purifying it by reverse-phase high-performance liquid chromatography to obtain a pure intestinal absorbable cholesterol-lowering peptide.

[0009] The use of the intestinal absorbable cholesterol-lowering peptide in the preparation of medicines or health products for preventing and / or treating hyperlipidemia or hepatobiliary diseases.

[0010] Use of the intestinal absorbable cholesterol-lowering peptide in the preparation of drugs or health products for preventing and / or treating coronary heart disease.

[0011] Beneficial effects:

[0012] Compared with the prior art, the intestinal absorbable cholesterol-lowering peptide of the present invention and its solid-phase synthesis method and application have the following advantages:

[0013] 1. The cholesterol-lowering pentapeptide of the present invention has a clear structure and can be prepared by solid-phase chemical synthesis or by separation and purification of whey protein hydrolysate. It has a wide source and reduces production costs.

[0014] 2. The cholesterol-lowering pentapeptide of the present invention has a good effect on lowering cholesterol content and has the potential to alleviate intestinal cholesterol absorption. It can be used to prepare drugs for hyperlipidemia, coronary heart disease, etc. or health products for preventing the occurrence of cholesterol-related diseases. It can also be used in combination with other health products or food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a process flow chart of the method for preparing the casein-derived cholesterol-lowering peptide of the present invention;

[0016] Figure 2 is the chromatogram of the peptide HTSGY (His-Thr-Ser-Gly-Tyr);

[0017] Figure 3 This is the MS / MS spectrum of the peptide HTSGY (His-Thr-Ser-Gly-Tyr);

[0018] Figure 4 Chemical cholesterol-lowering activity of HTSGY. DETAILED DESCRIPTION

[0019] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the implementation cases are conventional means well known to those skilled in the art. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0020] Example 1 Caco-2 cell culture

[0021] Fetal bovine serum (FBS) was added to DMEM (purchased from Boster Biosciences) at a 20% (v / v) concentration, followed by a 1% (v / v) double-antibody cocktail consisting of 100 U / mL penicillin and 100 μg / mL streptomycin. Caco-2 cells (purchased from Nanjing KeyGen Biotech Development Co., Ltd.) were grown in a cell culture incubator set at 37°C and 5% CO2. Medium changes and subcultures were performed promptly based on cell growth.

[0022] Example 2 Method for Isolating and Purifying His-Thr-Ser-Gly-Tyr Polypeptide

[0023] (1) Preparation of whey protein hydrolysate

[0024] A 6% whey protein hydrolysate (whey protein powder used, model WPC-80, manufactured by Hilmar Cheese Company, USA) was prepared with double-distilled water and heated at 65°C with stirring to fully dissolve it. The pH was adjusted to 8.5 with 0.5M NaOH and 0.5M HCl solution. The reaction apparatus was placed in a 50°C constant-temperature shaking water bath and 3% chymotrypsin was added for hydrolysis. During the enzymatic hydrolysis, 0.1 mol / L NaOH was continuously added to maintain a constant pH value of the solution. The reaction time was 5 h. After the reaction, the casein hydrolysate was heated at 90°C for 20 min to inactivate the enzyme and centrifuged at 7000×g for 10 min to obtain the supernatant.

[0025] (2) Preparation of whey protein peptide ultrafiltrate

[0026] The supernatant collected in step (1) is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da. The ultrafiltration membrane pressure is adjusted to 0.5 MPa (the pressure should not be too high to avoid damaging the instrument) and the flow rate is about 5-10 mL / min (a flow rate that is too fast will cause high pressure and damage the membrane structure, while a flow rate that is too slow will result in low efficiency) to obtain an ultrafiltration polypeptide solution with a molecular weight of less than 3 kDa. The ultrafiltrate is concentrated by vacuum freeze drying;

[0027] (3) Preparation of whey protein-derived crude peptides

[0028] The concentrated whey protein peptide ultrafiltrate was separated and purified by gel chromatography. The Sephadex G-10 solid was weighed and swelled with boiling water for 2 hours. A small amount of distilled water was added to the chromatography column (10*500mm) to exclude the air at the bottom of the chromatography column. The swollen Sephadex G-10 gel was shaken evenly and the gel was carefully introduced into the chromatography column with a glass rod. After the column was filled, it was fully balanced with distilled water. The ultrafiltrate was dissolved with distilled water to a mass concentration of 80 mg / mL, the sample volume was 3 ml, the elution rate was 1.5 ml / min, the UV detector wavelength was 220 nm, and the components were collected and freeze-dried.

[0029] (4) Caco-2 cell model simulates small intestinal absorption

[0030] The Caco-2 cells cultured in the example were seeded into the TransWell chamber at a seeding density of 2×10 5cells / mL, then add 0.5mL of cell suspension to the AP side (Transwell plate chamber) and 1.5mL of culture medium to the BL side (Tranwell plate chamber), and culture in a 37℃, 5% CO2 incubator. Change the culture medium every other day during the first week of the culture cycle, and change the medium every day thereafter. The culture cycle is 21 days. The two peak substances collected in step (3) are respectively prepared into 20 mg / mL active peptide solutions with HANKS buffer, and 0.5mL of casein peptide solution prepared with HANKS buffer is added to the AP side of the transwell chamber, and 1.5mL of HANKS buffer is added to the BL side. The Transwell plate is placed in a 37℃, 5% CO2 incubator for 1-2 hours. After the transfer is completed, the transferred sample is taken out from the BL side, enriched, filtered through a 0.22μm water filter membrane, and freeze-dried. The freeze-dried powder is collected for later use.

[0031] (5) Whey protein source peptide sequence analysis

[0032] The whey protein-derived peptides after transport through Caco-2 cells were selected and analyzed by UPLC-MS. The UPLC-MS analysis conditions were as follows:

[0033] Liquid chromatography analytical column: ACQUITY UPLC BEH130 C18 column (2.1×150 nm, 1.7 μm) (Waters, USA); mobile phase A: 100% acetonitrile; mobile phase B: 0.1% formic acid in water; injection volume: 8 μL; detection wavelength: 204 nm; flow rate: 0.3 mL / min; column temperature: 45°C; elution program: 0–1 min, mobile phase A 2%, mobile phase B 95%; 1–10 min, mobile phase A 30%, mobile phase B 60%; 10–17 min, mobile phase A 100%, mobile phase B 0%; 17–22 min, mobile phase A 2%, mobile phase B 95%;

[0034] The mass spectrometer used was a WATERS MALDI SYNAPT Q-TOF MS (Waters, USA), with nitrogen as the drying and nebulizing gas. The relevant mass spectrometry conditions were: ionization mode: ESI+; capillary voltage: 3.5 kVolts; cone voltage: 30 Volts; desolvation gas temperature: 300°C; ion source temperature: 100°C; desolvation gas flow rate: 500 lit / hr; cone gas flow rate (L / Hr): 50 lit / hr; collision energy: 6 / 35 Volts; mass range: 100-1500 m / z; detection voltage: 1600-1700 Volts. Amino acid sequence analysis of whey protein peptides was performed using Masslynx 4.1 software, combined with manual calculation of the peptide amino acid sequence, to identify whey protein peptide sequences that can be successfully transported from the small intestine to the blood in their intact form.

[0035] Figure 2 This is the MS / MS spectrum of the peptide HTSGY (His-Thr-Ser-Gly-Tyr).

[0036] Example 3 Synthesis of His-Thr-Ser-Gly-Tyr Peptide by Solid Phase Synthesis

[0037] The dichlororesin was swollen and washed, and the Fmoc protecting group was removed before amino acids were added for condensation. The removal-protection-condensation process was repeated until all amino acids were connected. The resin was cleaved to obtain a crude His-Thr-Ser-Gly-Tyr polypeptide, which was purified by reverse-phase high-performance liquid chromatography to obtain a pure HTSGY polypeptide (>90%).

[0038] Example 4 Study on the Chemical Cholesterol-Lowering Activity of Pentapeptide LHSMK

[0039] Cholesterol micelles were prepared by dissolving 10 mmol / L sodium taurocholate, 2 mmol / L cholesterol, 5 mmol / L oleic acid, and 135 mmol / L NaCl in PBS. The sample and blank groups were shaken homogenously, then ultrasonically emulsified for 1 hour, incubated at 37°C for 24 hours, and centrifuged at 8000 rpm for 10 minutes. The supernatant was then collected for later use.

[0040] The cholesterol content was determined according to the instructions of the total cholesterol (T-CHO) test kit (purchased from Nanjing Jiancheng Bioengineering Institute), and the OD was detected using a microplate reader at a wavelength of 510 nm.

[0041] The sample group consisted of 1 mL of pentapeptide solution and 4 mL of cholesterol micelles;

[0042] The blank group consisted of 1 mL of distilled water and 4 mL of cholesterol micelles;

[0043]

[0044] Figure 4 This is a test of the cholesterol degradation ability of HTSGY. ALPM and AVFK are also intestinal absorbable polypeptides, but their cholesterol-lowering abilities are significantly lower than that of the pentapeptide HTSGY.

[0045] In summary, the pentapeptide HTSGY of the present invention not only has the ability to lower cholesterol, but is also an intestinal absorbable polypeptide, and its cholesterol-lowering ability is higher than that of ALPM and AVFK.

[0046] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. Use of an intestinal absorbable cholesterol-lowering peptide in the preparation of a cholesterol-lowering drug, characterized in that: The amino acid sequence of the cholesterol-lowering peptide is His-Thr-Ser-Gly-Tyr, and the cholesterol-lowering peptide can be absorbed by the intestine.

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