Polypeptide with function of inhibiting cholesterol esterase activity as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide with a function of inhibiting cholesterol esterase activity as well as a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO.1-2. The two kinds of functional polypeptides obtained through screening are derived from natural plant soybeans and have a definite cholesterol lowering effect, the action mechanism of the polypeptides is that the activity of cholesterol esterase is specifically inhibited, hydrolysis and absorption of dietary cholesterol ester are reduced, then the cholesterol level in the body is reduced, and the cholesterol lowering effect is achieved. The action is doubly verified through a molecular docking technology and an in-vitro enzyme activity inhibition experiment, the mechanism is clear, and the action target is definite. In view of the characteristics, the two polypeptides as natural cholesterol-lowering active components can be directly used as functional factors to develop functional food and products in the related medicine field, which are beneficial to maintaining the health level of blood fat (cholesterol), and have remarkable application value and wide market prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to polypeptides with the function of inhibiting cholesterol esterase activity, their preparation methods, and applications. Background Technology

[0002] High cholesterol levels have become a significant risk factor threatening human health, and are significantly associated with the development of various chronic diseases such as coronary heart disease, malignant tumors, metabolic syndrome, and type 2 diabetes. Cholesterol esterase (CE), a key enzyme regulating cholesterol metabolism, is secreted by the pancreas into the duodenum. It catalyzes the hydrolysis of cholesterol esters into free cholesterol and fatty acids, which are easily absorbed by the intestinal mucosa, inducing hypercholesterolemia. Therefore, inhibiting CE activity to reduce dietary cholesterol absorption has become a new strategy for prevention and treatment. Statins are commonly used clinically to lower cholesterol, and although they are effective, they have drawbacks such as high treatment costs and potential adverse reactions. Therefore, the development of safe and effective natural cholesterol-lowering agents is essential.

[0003] From the perspective of nutritional intervention and functional food development, the bioactivity of plant-derived proteins and their enzymatic hydrolysates has attracted much attention. Bioactive peptides produced by dietary protein hydrolysis can regulate lipid metabolism and lower cholesterol. This invention aims to systematically screen and identify functional peptides with CE inhibitory activity after digestion of soybean peptide gel loaded with phytosterols using high-performance liquid chromatography-mass spectrometry sequencing, peptidomics analysis, and molecular docking, and to evaluate their cholesterol-lowering efficacy. This will provide a scientific basis for the development of novel functional foods that help maintain healthy blood lipid (cholesterol) levels, and fill the research gap in the discovery and verification of specific cholesterol-lowering peptides from soybean peptide nanogel digestion products. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide with the function of inhibiting cholesterol esterase activity, its preparation method, and its applications, thereby solving the problems existing in the prior art. The polypeptide provided by this invention has the function of inhibiting cholesterol esterase activity and can be directly used as a functional factor to develop functional foods and related pharmaceutical products that help maintain healthy blood lipid (cholesterol) levels, with significant application value and broad market prospects.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a polypeptide with the function of inhibiting cholesterol esterase activity, wherein the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO.1-2.

[0007] The present invention also provides a method for preparing the above-mentioned polypeptide, comprising the step of preparing the polypeptide by solid-phase synthesis.

[0008] The present invention also provides the application of the above-mentioned polypeptide in the preparation of cholesterol-lowering drugs.

[0009] The present invention also provides a cholesterol-lowering drug, the active ingredient of which includes the above-mentioned polypeptide.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the excipients include fillers, excipients, binders, disintegrants, emulsifiers, or preservatives.

[0012] The present invention also provides the application of the above-mentioned polypeptides in the preparation of functional foods that help maintain healthy blood lipid levels.

[0013] The present invention also provides a functional food that helps maintain healthy blood lipid levels, the active ingredients of which include the aforementioned polypeptides.

[0014] The present invention also provides the use of the above-mentioned polypeptide in the preparation of cholesterol esterase inhibitors.

[0015] The present invention also provides a cholesterol esterase inhibitor, the active ingredient of which includes the above-mentioned polypeptide.

[0016] The present invention discloses the following technical effects:

[0017] This invention, through systematic research on the digestive and metabolic processes of soybean peptide nanogels, successfully screened and prepared functional peptides HLPSYSPYPQ and SSPDIFNPQ with highly efficient cholesterol esterase (CE) inhibitory activity. These peptides significantly reduce intestinal absorption by specifically inhibiting cholesterol esterase activity, blocking the hydrolysis of cholesterol esters in the diet, thereby lowering cholesterol levels in the body from the source. This mechanism of action was verified by both molecular docking simulation and in vitro enzyme activity inhibition experiments, demonstrating a clear mechanism and well-defined targeting.

[0018] Given the above characteristics, these two peptides, as natural cholesterol-lowering active ingredients, can be directly used as functional factors to develop functional foods and related pharmaceutical products that help maintain healthy blood lipid (cholesterol) levels, and have significant application value and broad market prospects.

[0019] Furthermore, this invention establishes a multi-dimensional screening system guided by cholesterol esterase inhibitory activity, integrating mass spectrometry sequencing, molecular docking, and bioactivity verification technologies. This system overcomes the bottlenecks of low efficiency and ambiguous target sites in traditional functional peptide screening, providing a standardized technical path for the precise development of natural cholesterol-lowering peptides. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The diagram shows the molecular docking of simvastatin and CE; where A is the relative overall structure diagram of simvastatin and CE; B is the two-dimensional interaction diagram of simvastatin and CE; and C is the force type diagram of simvastatin and CE.

[0022] Figure 2 The diagram shows the molecular docking of HQ and CE; where A is the relative overall structure diagram of HQ and CE; B is the two-dimensional interaction diagram of HQ and CE; and C is the force type diagram of HQ and CE.

[0023] Figure 3 The diagram shows the molecular docking of SQ and CE; where A is the relative overall structure diagram of SQ and CE; B is the two-dimensional interaction diagram of SQ and CE; and C is the force type diagram of SQ and CE.

[0024] Figure 4 The graph shows the relationship between HQ peptide concentration and cholesterol esterase inhibition.

[0025] Figure 5 This is a graph showing the relationship between SQ peptide concentration and cholesterol esterase inhibition. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Example 1

[0032] 1. Materials and Methods

[0033] 1.1 Materials

[0034] Soy protein isolate (protein content ≥95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China); phytosterols were purchased from Shaanxi Haisifu Biotechnology Co., Ltd. (Shaanxi, China); alkaline protease (400,000 U / g) was purchased from Angel Enzyme Preparations Co., Ltd. (Yichang, China); pepsin (4,000 U / mL) and trypsin (200 U / mL) were purchased from Xiaodong Yijian Instrument Co., Ltd. (Suzhou, China); all other reagents were analytical grade.

[0035] 1.2 Preparation and digestion of nanogels

[0036] Alkaline protease (enzyme to substrate ratio 1:10) was added to a 5 wt% soybean protein aqueous solution and hydrolyzed at 55 °C for 1 hour to obtain soybean peptides with a degree of hydrolysis of 5.21%. The enzyme was inactivated by heating at 100 °C for 15 minutes, and the supernatant was collected by centrifugation at 8000 r / min for 15 minutes. 22.5 mg of phytosterols were encapsulated in 50 mL of soybean peptides using an emulsion evaporation method to obtain LSPN nanogels.

[0037] Alkaline protease (enzyme to substrate ratio 1:10) was added to a 5 wt% soybean protein aqueous solution and hydrolyzed at 55 °C for 3 hours to obtain soybean peptides with a degree of hydrolysis of 15.96%. The enzyme was inactivated by heating at 100 °C for 15 minutes, and the supernatant was collected by centrifugation at 8000 r / min for 15 minutes. 22.5 mg of phytosterol was encapsulated in 50 mL of soybean peptides using an emulsion evaporation method to obtain MSPN nanogel.

[0038] Simulated gastric juice (SGF) and simulated intestinal juice (SIF) were prepared according to the method described in the literature “Gao, Y., Chen, L., Chi, H., Li, L., & Teng, F. (2024). Insights into the soybean protein isolate hydrolysates: Performance characterization, emulsion construction and invitro digestive behavior. International Journal of Biological Macromolecules, 279(3), 135372”.

[0039] Gastric digestion: Adjust the pH of SPN to 2.0, mix with an equal amount of SGF, shake at 37°C for 2 hours, heat at 100°C for 10 minutes to inactivate the enzyme, centrifuge at 4000 rpm for 10 minutes and collect the supernatant to obtain the gastric digestion product.

[0040] Intestinal digestion: Adjust the pH of the gastric digestion products to 7.0 with 0.1 mol / L NaOH, add equal amounts of SIF, bile salts and pancreatic enzymes, shake at 37°C for 3 hours, heat at 100°C for 10 minutes to terminate the reaction, centrifuge at 4000 rpm for 15 minutes and collect the supernatant, which is the intestinal digestion product.

[0041] 1.3 Peptide Identification and Screening

[0042] After desalting the enteric digestion products of LSPN and MSPN using a C18 column, the amino acid sequences were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), and high-frequency peptides were obtained by searching the UniProtGlycinemax database.

[0043] 1.4 Molecular docking

[0044] The three-dimensional structures of the screened peptides were constructed in ChemBioDraw Ultra 22.0, and docked with CE (PDBID: 1F6W) using Autodock Vina 1.5.6. The conformation with the lowest binding energy was selected as the optimal conformation. The interaction forces were analyzed and visualized using Discovery Studio 4.5 Client.

[0045] 1.5 Peptide Synthesis and Inhibition Experiments

[0046] The screened peptides were synthesized using a solid-phase synthesis method, as follows:

[0047] First, weigh 0.5 g of 2-chlorotriphenylmethyl resin and swell it in DCM for 30 minutes. Then, add 3 molar excess of Fmoc-Asn(Trt)-OH and 10 molar excess of DIEA, dissolve in a small amount of DMF, and shake for 1 hour to inoculate the first amino acid. Next, wash 6 times alternately with DMF and DCM. Then, deprotect with 20% piperidine DMF solution (5 minutes first, then 15 minutes). After removing the solution, take the resin and wash it with ethanol. The test with ninhydrin and other solutions showed a positive result. Then, wash sequentially with DMF, methanol, and DMF. Next, add 3 molar excess of the protected amino acid (such as Fmoc-Ala-OH) and HBTU, and 10 molar excess of DIEA for condensation for 40 minutes. After washing, repeat the deprotection, detection, washing, and condensation steps, linking all amino acids in the sequence from right to left. Finally, the test was negative, and the resin was washed with methanol. Next, a cleavage solution was prepared using TFA, TIS, EDT, and H2O in a specific ratio. This solution was mixed with the resin at a ratio of 10 mL / g and kept at a constant temperature with shaking for 2 hours for cleavage. The lysis buffer was dried under nitrogen, then chromatographically analyzed with diethyl ether and washed six times. The resulting crude peptide was evaporated at room temperature. 200 mg of the crude peptide was then dissolved in a 50% acetonitrile aqueous solution, sonicated, filtered, and analyzed by analytical-grade HPLC and preparative-grade HPLC. Purity was determined by MS. The purified solution was lyophilized to obtain the final product, which was then sealed and stored at -20°C.

[0048] The purity of the product was determined to be ≥95% by reversed-phase high-performance liquid chromatography, and cholesterol esterase inhibition experiments could be performed.

[0049] Cholesterol esterase inhibition assay: The reaction system contained 50 μL of 10 mM p-nitrophenylbutyrate (dissolved in 5.16 mM sodium taurocholate and 100 mM NaCl phosphate buffer), 50 μL of peptide solutions of different concentrations, and 50 μL of 25 μg / mL CE solution. After incubation at 37 °C for 5 min, the absorbance was measured at 405 nm and the inhibition rate was calculated.

[0050] CE inhibition rate = [1 - (CD) / (AB)] × 100%;

[0051] Where A is the absorbance value with enzyme but no sample, B is the absorbance value with neither sample nor enzyme, C is the absorbance value with both sample and enzyme, and D is the absorbance value with neither sample nor enzyme.

[0052] Dosage effect verification: Prepare peptide solutions of different concentrations and determine the inhibition rate according to the cholesterol esterase inhibition experiment method described above.

[0053] 1.6 Statistical Analysis

[0054] The experiment was repeated three times. The results are expressed as mean ± standard deviation. One-way ANOVA and Duncan's test were used to evaluate the results. p < 0.05 was considered statistically significant.

[0055] 2. Experimental Results

[0056] 2.1 Results of peptide screening

[0057] The LSPN enteric digestion products were desalted, analyzed by HPLC-MS / MS, and searched by database to obtain the peptide HLPSYSPYPQ (named HQ; SEQ ID NO.1); the MSPN enteric digestion products were desalted, analyzed by HPLC-MS / MS, and searched by database to obtain the peptide SSPDIFNPQ (named SQ; SEQ ID NO.2).

[0058] As shown in Table 1, the bioactivity of HQ was assessed using PeptideRanker, with a score of 0.514632. The probability of its interaction with cholesterol esterase (CE, PDBID: 1F6W) was predicted using PepSite2, with a p-value of 0.009973, meeting the screening criteria for bioactive peptides. SQ had a PeptideRanker score of 0.71451, and the p-value for its interaction with CE predicted by PepSite2 was 0.02882, indicating that SQ is a high-potential bioactive peptide.

[0059] 2.2 Results of Molecular Docking Experiment

[0060] The three-dimensional structure of HQ was constructed in ChemBioDraw Ultra 22.0, and docked with CE (PDBID: 1F6W) using Autodock Vina 1.5.6. The conformation with the lowest binding energy was selected as the optimal conformation. The interaction forces were analyzed and visualized using Discovery Studio 4.5 Client. The docking binding energy between HQ and CE was -7.0 kcal / mol, and there were 10 binding sites, including VAL366, ALA365, and TYR367. Among them, HQ formed a strong hydrogen bond interaction at VAL366, which directly occupied the substrate binding site near the active site of CE, hindering the binding of cholesterol ester to the enzyme. At the same time, a Pi-alkyl hydrophobic interaction was formed at LEU394, which further stabilized the conformation of the peptide-enzyme complex, allowing HQ to bind more firmly to CE, thereby weakening the catalytic hydrolysis ability of CE on cholesterol ester. Figure 2 ).

[0061] In ChemBioDraw Ultra 22.0, a 3D structure of SQ was constructed and docked with CE (PDBID: 1F6W). The conformation with the lowest binding energy was then selected. For example... Figure 3As shown, the docking energy between SQ and CE is -8.7 kcal / mol (strongest affinity), and the binding sites include LEU224, PRO226, ILE229, LYS231, TRP236, and HIS283. The aspartic acid residues of SQ form conventional hydrogen bonds with LYS231, which helps the SQ peptide precisely locate to the edge of the active pocket of CE. Simultaneously, isoleucine residues form Alkyl hydrophobic interactions with ILE229 and LEU224, and phenylalanine residues form Pi-Pi stacking interactions with TRP236. These multiple interactions stably embed SQ into the hydrophobic cavity of CE. Importantly, SQ also binds to simvastatin at sites such as ILE399 and LEU527. Figure 1 The binding of SQ overlaps with the catalytic center Ser194, and these sites are key regions for CE-catalyzed cholesterol ester hydrolysis. The binding of SQ directly interferes with the enzyme's catalytic conformation, preventing the substrate from contacting the catalytic center Ser194, thereby significantly inhibiting CE activity.

[0062] Table 1. Bioactivity prediction and molecular docking results of key peptides

[0063]

[0064] 2.3 Inhibition Experiment Results

[0065] The results of the cholesterol esterase inhibition assay showed that the inhibition rate of CE by 20 mg / mL HQ solution was 26.10%; and the inhibition rate of CE by 10 mg / mL SQ solution was 44.67%.

[0066] Prepare 0.75-40 mg / mL HQ solution and perform the cholesterol esterase inhibition assay according to the prescribed method. The results are shown in [Figure number missing]. Figure 4 .Depend on Figure 4 It was found that within the HQ concentration range of 0.75-20 mg / mL, the inhibition rate of HQ on cholesterol esterase increased rapidly with increasing concentration, from 1.87% to 26.10%. However, when the peptide concentration exceeded 20 mg / mL, the inhibition rate plateaued, remaining at around 26%. This indicates that HQ achieves a good inhibitory effect at around 20 mg / mL and tends to stabilize thereafter, with subsequent increases in concentration having limited effect on improving the inhibition rate.

[0067] Prepare a 0.375-40 mg / mL SQ solution and perform the cholesterol esterase inhibition assay. Results are shown below. Figure 5As shown in Figure 5, in the peptide concentration range of 0.375-20 mg / mL, the inhibition rate increased with increasing concentration, jumping from 3.33% to 60.72%. When the peptide concentration reached 20 mg / mL, the inhibition rate plateaued, remaining in the 60%-62% range. This indicates that SQ's inhibitory effect on CE is saturated at around 20 mg / mL, and subsequent increases in concentration did not significantly change the inhibitory effect, demonstrating SQ's highly efficient CE inhibition capability.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A polypeptide with the function of inhibiting cholesterol esterase activity, characterized in that, The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO.1-2.

2. A method for preparing the polypeptide as described in claim 1, characterized in that, The method includes the step of preparing the polypeptide using a solid-phase synthesis method.

3. The use of the polypeptide as described in claim 1 in the preparation of cholesterol-lowering drugs.

4. A cholesterol-lowering drug, characterized in that, The active ingredient includes the polypeptide described in claim 1.

5. The cholesterol-lowering drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The cholesterol-lowering drug according to claim 5, characterized in that, The excipients include fillers, excipients, binders, disintegrants, emulsifiers, or preservatives.

7. The use of the polypeptide as described in claim 1 in the preparation of functional foods that help maintain healthy blood lipid levels.

8. A functional food that helps maintain healthy blood lipid levels, characterized in that, The active ingredient includes the polypeptide described in claim 1.

9. The use of the polypeptide as described in claim 1 in the preparation of a cholesterol esterase inhibitor.

10. A cholesterol esterase inhibitor, characterized in that, The active ingredient includes the polypeptide described in claim 1.

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