A yolk-derived peptide for lowering blood lipids, a preparation method, a pharmaceutical composition and use thereof

Abstract

This invention relates to the pharmaceutical field, specifically to an egg yolk-derived peptide for lowering blood lipids, its preparation method, pharmaceutical composition, and uses. This invention discovers an egg yolk-derived peptide obtained by enzymatic hydrolysis, digestion, separation, and purification of defatted egg yolk powder. Its amino acid sequence is shown in SEQ ID NO.1. This peptide possesses high potential activity for lowering blood lipids. In vitro pancreatic lipase inhibition experiments and hyperlipidemic cell model experiments demonstrate that the egg yolk-derived peptide of this invention can affect lipid absorption and metabolism, thereby alleviating lipid metabolism disorders and lipid accumulation, ultimately reducing TC and TG levels.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to an egg yolk-derived peptide for lowering blood lipids, its preparation method, pharmaceutical composition, and uses. Background Technology

[0002] Cardiovascular disease (CVD) is the leading cause of death worldwide, and its underlying pathology, atherosclerosis, is directly related to lipid metabolism disorders, particularly hyperlipidemia. Hyperlipidemia is characterized by abnormally elevated levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) in plasma, as well as decreased levels of high-density lipoprotein cholesterol (HDL-C). According to the World Health Organization, hundreds of millions of people worldwide are affected by hyperlipidemia. It is not only an independent risk factor for acute cardiovascular and cerebrovascular events such as myocardial infarction and stroke, but is also closely associated with metabolic diseases such as hypertension, diabetes, and non-alcoholic fatty liver disease, constituting a significant public health and economic burden.

[0003] Currently, the mainstream lipid-lowering drugs in clinical practice mainly include statins, fibrates, cholesterol absorption inhibitors (such as ezetimibe), and novel PCSK9 inhibitors. Among them, statins, as first-line drugs, significantly reduce LDL-C levels by inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis in hepatocytes, and have established a core position in the primary and secondary prevention of cardiovascular events. However, long-term or high-dose use of statins may be accompanied by a series of problems: some patients experience muscle pain, myositis, or even rhabdomyolysis and other muscle toxicities; there is a risk of abnormal liver function; in addition, a considerable proportion of patients cannot achieve their ideal blood lipid targets with statin treatment, i.e., there is a clinical dilemma of "statin intolerance" or "statin inadequacy." Other drugs also have their limitations, such as the effects of fibrates on liver and kidney function, and the increased risk of myopathy when used in combination with statins; PCSK9 inhibitors, although potent, are expensive, require subcutaneous injection, and have limited accessibility. Therefore, developing novel lipid-lowering functional factors with novel mechanisms of action, high safety, natural sources, and controllable costs has become an important research direction in the fields of nutrition and pharmacy.

[0004] Bioactive peptides are a class of small molecule compounds composed of amino acids linked by peptide bonds, possessing specific physiological regulatory functions. Among them, lipid-lowering peptides have attracted widespread attention because they can be ingested through diet or supplements and intervene in lipid metabolism in a multi-target, mild regulatory manner. Compared with chemically synthesized drugs, lipid-lowering peptides are usually derived from food proteins (such as milk protein, soy protein, and marine fish protein), exhibiting better biocompatibility and safety expectations, and are less likely to accumulate in the body and produce toxicity. Active peptides in quinoa protein hydrolysate have excellent pancreatic lipase inhibitory activity and reduce TG levels in 3T3-L1 cells. Similarly, active peptides with PL inhibitory activity have been identified in sea cucumber collagen-derived peptides, oyster-derived peptides, and spirulina-derived peptides, showing potential for lipid regulation. Furthermore, chickpea peptide VFVRN, in the C57BL / 6J high-fat animal model and 3T3-L1 cell model, regulates the activity of lipid metabolism enzymes through the AMPK-AKT1 / PPARG pathway to control lipid metabolism. Tea protein-derived lipid-lowering peptides exhibit excellent pancreatic lipase inhibitory activity. Barracuda skin collagen peptides can reduce TC, TG, and LDL-C levels in a high-fat zebrafish model. Rapeseed protein-derived peptides enhance LDL-C uptake and clearance in 3T3-L1 and HepG2 cells via the LDLR-PCSK9 pathway, thereby reducing TC levels. Therefore, the discovery and development of novel, efficient, and safe lipid-lowering peptides, along with enhancing their stability and bioavailability, has significant scientific value and application prospects for enriching lipid-lowering treatment methods. Summary of the Invention

[0005] This invention provides an egg yolk-derived peptide for lowering blood lipids, its preparation method, pharmaceutical composition, and uses.

[0006] The present invention provides an egg yolk-derived peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for preparing an egg yolk-derived peptide with an amino acid sequence as shown in SEQ ID NO.1, comprising the following steps: The defatted egg yolk powder is enzymatically hydrolyzed to obtain defatted egg yolk powder hydrolysate. The defatted egg yolk powder hydrolysate is then subjected to in vitro simulated gastrointestinal digestion. The digestion products are then separated and purified to obtain the final product.

[0008] Preferably, the enzymatic hydrolysis includes the following steps: adding water and trypsin to the defatted egg yolk powder, adjusting the pH of the solution, and then performing enzymatic hydrolysis.

[0009] Preferably, the ratio of the defatted egg yolk powder to water is 1 part by weight: 5-20 parts by volume, and the ratio of parts by weight to parts by volume is 1 part by weight: 1 part by volume = 1 g: 1 mL; the amount of trypsin added is 6000-12000 U enzyme activity / g defatted egg yolk powder; the pH of the solution is adjusted to 7.5-8.5 every hour during enzymatic hydrolysis; and the enzymatic hydrolysis time is 2-6 hours.

[0010] Preferably, the ratio of the defatted egg yolk powder to water is 1 part by weight: 10 parts by volume, and the ratio of parts by weight to parts by volume is 1 part by weight: 1 part by volume = 1 g: 1 mL; the amount of trypsin added is 10000 U enzyme activity / g defatted egg yolk powder; the pH of the solution is 8.0; and the enzymatic hydrolysis time is 4 hours.

[0011] Preferably, the enzymatic hydrolysis further includes the following steps: The enzyme hydrolysate was inactivated by boiling in a water bath for 10 min, then cooled to 25°C and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was collected to obtain the deoiled egg yolk powder hydrolysate.

[0012] The present invention also provides the use of egg yolk peptides with amino acid sequences as shown in SEQ ID NO.1 in the preparation of lipid-lowering drugs.

[0013] The present invention also provides a pharmaceutical composition for lowering blood lipids, which is made by adding pharmaceutically acceptable excipients or auxiliary ingredients, with an egg yolk peptide having an amino acid sequence as shown in SEQ ID NO.1 as the active ingredient.

[0014] The egg yolk-derived peptide His-Gly-Phe-Trp-Pro-Arg of this invention exhibits high potential activity in lowering blood lipids. In vitro pancreatic lipase inhibition experiments and hyperlipidemic cell model experiments show that the hexapeptide His-Gly-Phe-Trp-Pro-Arg can affect lipid absorption and metabolism, thereby alleviating lipid metabolism disorders and lipid accumulation, and ultimately reducing TC and TG levels.

[0015] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0016] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0017] Figure 1 The total ion chromatogram of the sample after simulated gastrointestinal digestion of degreased egg yolk powder hydrolysate was determined by mass spectrometry.

[0018] Figure 2 This is the secondary mass spectrum of the hexapeptide His-Gly-Phe-Trp-Pro-Arg.

[0019] Figure 3 This image shows the docking results of the hexapeptide His-Gly-Phe-Trp-Pro-Arg with pancreatic lipase.

[0020] Figure 4 The in vitro pancreatic lipase inhibitory activity of defatted egg yolk powder hydrolysate and hexapeptide His-Gly-Phe-Trp-Pro-Arg is shown in the figure.

[0021] Figure 5 Oil Red staining images of HepG2 cells in the blank control group (A), high-fat model group (B), and polypeptide treatment group (C), and a statistical graph of the Oil Red staining area of ​​HepG2 cells in the blank control group, high-fat model group, and polypeptide treatment group (D).

[0022] Figure 6 This is a statistical graph showing the TG and TC levels in HepG2 cells from the blank control group, high-fat model group, and peptide treatment group. The same letter in the graph indicates no significant difference. P >0.05), different letters indicate significant differences ( P <0.05). Detailed Implementation

[0023] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0024] Example 1: Preparation and identification of egg yolk-derived peptides with sequences as shown in SEQ ID NO.1 The amino acid sequence (SEQ ID NO.1) of the egg yolk-derived peptide is as follows: HGFWPR.

[0025] Using defatted egg yolk powder as raw material (defatted egg yolk powder is a product obtained by removing triglycerides and cholesterol from whole egg powder through supercritical CO2 treatment), a certain volume of double-distilled water was added (egg yolk powder: water = 1:10), and trypsin was added (enzyme dosage of 10000 U enzyme activity / g defatted egg yolk powder). The pH of the solution was adjusted to the suitable pH value for trypsin, and after mixing, it was placed in a constant temperature shaking water bath with the suitable enzyme temperature set for enzymatic hydrolysis. The enzymatic hydrolysis time was 4 hours, and the pH was adjusted to 8.0 every hour during the enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling water bath for 10 min, then cooled to room temperature and centrifuged at 8000 rpm for 10 min at 4℃. The supernatant was collected to obtain the defatted egg yolk powder hydrolysate. After freeze-drying, it was digested in vitro in the gastrointestinal tract. The supernatant of the digestion solution was centrifuged at 8000 rpm for 10 min, the supernatant was collected, and freeze-dried for later use to obtain the digestion product.

[0026] The lyophilized digestion product sample was dissolved in mobile phase A to prepare a 0.5 mg / mL sample solution. 1 μL of the sample solution was injected into the analytical column (C18 capillary column). Mobile phase A: 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid, flow rate 300 nL / min. After chromatographic separation, the sample was analyzed by mass spectrometry using a Tims TOF Pro mass spectrometer. The total ion chromatogram is shown below. Figure 1 Based on the LC-MS / MS secondary mass spectrum ( Figure 2 For the hexapeptide HGFWPR, the complete γ ion series (γ1) was detected. + ~y5 + The m / z values ​​are 175.11895, 272.17172, 458.25103, 605.31944, and 662.34091, respectively. Fragment peaks are present from y1 to y5, supporting the sequential ligation of the C-terminal sequence RPWFGH) and the b-ion series (b1). + ~b5 + The m / z values ​​were 138.06619, 195.08765, 342.15607, 528.23538, and 625.28814, respectively. Only b2 was detected. + and b3 + And b1 + b4 + and b5 + Undetectable. This may be related to the effect of proline residues during fragmentation—the presence of proline (P) tends to inhibit the breaking of its amino-terminal peptide bond, leading to reduced yields of b4 (located between WP) and b5 (located between PR). Furthermore, b1... + As the smallest fragment ion, it has a low mass-to-charge ratio, is easily affected by background ion interference, and has insufficient signal strength. Although the b-ion series is incomplete, the complete g-ion series (y1) is... + ~y5+ All γ-ion sequences were detected and highly matched the theoretical values, sufficient to confirm that the amino acid sequence of the hexapeptide is HGFWPR. Therefore, the sequential linking of the N-terminal sequence HGFWP is supported. According to the fragmentation rules of peptide tandem mass spectrometry, although the b-ion series is incomplete, the detected complete γ-ion series and partial b-ion series are sufficient to confirm that the amino acid sequence of the hexapeptide is HGFWPR. This result indicates that the digested product sample contains the hexapeptide His-Gly-Phe-Trp-Pro-Arg. The Peptide Ranker predicts that His-Gly-Phe-Trp-Pro-Arg has an activity score of 0.97, ranking first among all peptides (Table 1), revealing its significant advantages in structural characteristics and biological activity potential. Furthermore, all peptides with Peptide Ranker scores above 0.5 did not show any potential toxicity risk (Table 2). All peptides were analyzed using the ADMET model. None of the peptides inhibited multiple key cytochrome P450 metabolic enzymes (including CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) (Table 3), indicating a low risk of interaction with commonly used drugs during in vivo metabolism and a significant advantage from a safety perspective. Molecular docking of the peptides with pancreatic lipase (PL) using Autodock Vina revealed that the hexapeptide His-Gly-Phe-Trp-Pro-Arg had the strongest binding energy (-8.4 kcal / mol), indicating its strongest PL inhibitory potential. The molecular docking results are shown in [Table 3]. Figure 3 .

[0027] Table 1. Peptide Ranker Rating Results Table 2. Results of Potential Toxicity Risk Assessment of Peptides Table 3. ADMET Model Analysis Results The following experimental examples illustrate the beneficial effects of the hexapeptide His-Gly-Phe-Trp-Pro-Arg.

[0028] Experimental Example 1: In vitro activity assay of active hexapeptide The pancreatic lipase (240 U / mg) used in this experiment was purchased from Shanghai Maclean Biotechnology Co., Ltd., and the substrate p-NPB was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (reaction concentration was 0.75 mM).

[0029] The hexapeptide His-Gly-Phe-Trp-Pro-Arg was prepared using solid-phase chemical synthesis, and its in vitro lipid-lowering activity was evaluated. A 2 mg / mL sample solution was prepared by reconstituted the hydrolysate and hexapeptide solution with 0.1 M Tris-HCl. The sample solution and pancreatic lipase (PL) solution (10 mg / mL) were added to 96-well plates and incubated at 37 °C for 10 min. Preheated p-NPB solution was then added, and the total volume was brought to 250 μL with Tris-HCl solution. Incubation was continued for another 20 min. After the reaction was complete, the enzyme was inactivated in boiling water for 10 min. The reaction solution was then filtered through a 0.22 μm microporous membrane, and the absorbance was measured at 405 nm. The specific amounts of each solution added are shown in the table below.

[0030] Table 4. Composition of each system in the pancreatic lipase inhibition experiment The results showed that the in vitro pancreatic lipase inhibition rate of the hydrolysate was 38.85 ± 4.57% (IC50). 50 The in vitro pancreatic lipase inhibition rate of the hexapeptide His-Gly-Phe-Trp-Pro-Arg was 70.66 ± 4.41% (3.85 ± 0.14 mg / mL). Figure 4 IC 50 The value was 1.36 ± 0.13 mg / mL (equivalent to 1.70 ± 0.16 mM), representing an 81.88% increase in inhibition rate compared to the hydrolysate. This result confirms that the key active peptide His-Gly-Phe-Trp-Pro-Arg, screened through molecular docking, has high potential for lowering blood lipids.

[0031] Experiment Example 2: Oil Red Staining Experiment of HepG2 Cells HepG2 cells were seeded in 6-well plates and cultured at 37°C in medium containing 10% fetal bovine serum (FBS) (10% FBS, 89% DMEM, and 1% penicillin-streptomycin). When confluence reached 70-80%, the original medium was discarded, and medium containing 2% FBS (2% FBS, 97% DMEM, and 1% penicillin-streptomycin) was added and incubated for 12 h. The peptide-treated group was transferred to serum-free DMEM and cultured for 24 h in 0.5 mM OA working solution (containing 200 μg / mL hexapeptide His-Gly-Phe-Trp-Pro-Arg). Cells were fixed, stained with Oil Red O and H&E according to the Oil Red O kit, and lipid droplet distribution and cell morphology were observed under an inverted microscope. After culturing in DMEM containing 2% fetal bovine serum for 12 h as described above, the blank control group was incubated in DMEM containing 1% BSA (fatty acid-free) for 24 h. The high-lipid model group was transferred to serum-free DMEM and cultured in 0.5 mM OA working solution for 24 h. The remaining experimental conditions were the same as those for the peptide treatment group. The lipid droplet staining area was analyzed using ImageJ software, and the results are presented as the ratio of Oil Red O staining area (representing lipid droplets) to H&E staining area (representing cells).

[0032] The results showed that a small number of lipid droplets were present in the blank control group, and the cell morphology was normal. In the oleic acid-induced high-lipid model group, the cells were filled with a large number of red lipid droplets, which were diffusely and clustered, and the cytoplasm was almost filled with lipid droplets. In contrast, the number and density of red lipid droplets in the polypeptide treatment group were significantly reduced, and the distribution of lipid droplets tended to be more dispersed. Figure 5 This indicates that the hexapeptide His-Gly-Phe-Trp-Pro-Arg can significantly alleviate oleic acid-induced lipid deposition in cells.

[0033] Experimental Example 3: HepG2 cell TC and TG levels HepG2 cells were seeded in 96-well plates and cultured at 37°C in medium containing 10% fetal bovine serum (10% fetal bovine serum, 89% DMEM and 1% penicillin-streptomycin). When the confluence reached 70-80%, the original medium was discarded and medium containing 2% fetal bovine serum (2% fetal bovine serum, 97% DMEM and 1% penicillin-streptomycin) was added and incubated for 12 h. The high-fat HepG2 cell model was induced by transferring the high-fat HepG2 cell group into serum-free DMEM and adding 0.5 mM oleic acid working solution. The peptide treatment group was transferred into serum-free DMEM and added 0.5 mM oleic acid working solution containing 200 μg / mL hexapeptide His-Gly-Phe-Trp-Pro-Arg. The blank control group was incubated in DMEM solution containing 1% BSA (fatty acid-free). After incubation for 24 h, the cells were washed twice with pre-cooled PBS, and 50 μL of RIPA cell lysis buffer was added to each well to dissolve the cells for 15 min. The lysates were collected and centrifuged at 12000 rpm for 10 min. The total protein content in the lysates was measured using a BCA protein assay kit (Beijing Biosharp Co., Ltd.). Subsequently, the levels of TG and TC in the lysates were measured by absorbance using a total cholesterol and triglyceride kit (Total Cholesterol and Triglyceride Kit, Nanjing Jiancheng Bioengineering Institute).

[0034] The results showed that the intracellular TG content in the blank control group was 0.0048 ± 0.0011 mmol / gprot, while the TG content in the oleic acid-induced high-fat model group significantly increased to 0.0169 ± 0.0015 mmol / gprot, approximately 3.5 times that of the control group. After intervention with the hexapeptide His-Gly-Phe-Trp-Pro-Arg, the TG content decreased to 0.00586 ± 0.0009 mmol / gprot, approaching that of the blank control group and decreasing by 65.3% compared to the model group. Furthermore, the TC detection results showed the same trend as TG. Figure 6 ).

[0035] In summary, the hexapeptide His-Gly-Phe-Trp-Pro-Arg derived from defatted egg yolk powder exhibits high potential activity in lowering blood lipids. Combined with in vitro PL inhibition activity experiments and hyperlipidemic cell model experiments, it is shown that the hexapeptide His-Gly-Phe-Trp-Pro-Arg can affect lipid absorption and metabolism, thereby alleviating lipid metabolism disorders and lipid accumulation, and ultimately reducing TC and TG levels.

Claims

1. A yolk-derived peptide, characterized in that, The amino acid sequence of the egg yolk peptide is shown in SEQ ID NO.

1.

2. A method for preparing an egg yolk-derived peptide having an amino acid sequence as set forth in SEQ ID NO. 1, characterized in that, Includes the following steps: The degreased egg yolk powder is enzymatically hydrolyzed to obtain degreased egg yolk powder hydrolysate. The degreased egg yolk powder hydrolysate is then subjected to in vitro simulated gastrointestinal digestion. The digestion products are then separated and purified to obtain the final product. The enzymatic hydrolysis includes the following steps: adding water and trypsin to the defatted egg yolk powder, adjusting the pH of the solution, and then carrying out enzymatic hydrolysis; The ratio of the defatted egg yolk powder to water is 1 part by weight: 5-20 parts by volume, and the ratio of parts by weight to parts by volume is 1 part by weight: 1 part by volume = 1 g: 1 mL; the amount of trypsin added is 6000-12000 U enzyme activity / g defatted egg yolk powder; the pH of the solution is adjusted to 7.5-8.5 every hour during enzymatic hydrolysis; the enzymatic hydrolysis time is 2-6 hours.

3. The production method according to claim 2, characterized by, The ratio of the defatted egg yolk powder to water is 1 part by weight: 10 parts by volume, and the ratio of parts by weight to parts by volume is 1 part by weight: 1 part by volume = 1 g: 1 mL; the amount of trypsin added is 10000 U enzyme activity / g defatted egg yolk powder; the pH of the solution is 8.0; and the enzymatic hydrolysis time is 4 hours.

4. The production method according to claim 2, characterized by, The enzymatic hydrolysis also includes the following steps: The enzyme hydrolysate was inactivated by boiling in a water bath for 10 min, then cooled to 25°C and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was collected to obtain the deoiled egg yolk powder hydrolysate.

5. Use of egg yolk peptides with amino acid sequences as shown in SEQ ID NO.1 in the preparation of lipid-lowering drugs.

6. A pharmaceutical composition for reducing blood lipids, characterized by, It is made with egg yolk peptides with amino acid sequences as shown in SEQ ID NO.1 as the active ingredient, and with the addition of pharmaceutically acceptable excipients or auxiliary ingredients.

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