Hypolipidemic fish oil polypeptide composition and application thereof

Abstract

The present application relates to a kind of fish oil polypeptide composition for reducing blood fat and its application, belong to the field of biological medicine and functional food.The composition includes fish oil and pumpkin seed source active peptide C2-1 (molecular weight <3kDa), wherein active peptide accounts for 1-5% of the total weight of composition.Its preparation method includes: pumpkin seed protein complex zymolysis, ultrafiltration separation, anion exchange chromatography purification obtains C2-1 active peptide (KRGGRFL), then with fish oil homogenization into stable dispersion.The composition significantly reduces serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) by simultaneously inhibiting cholesterol esterase and lipase activity, and improves high-density lipoprotein cholesterol (HDL-C).Animal experiments show that its lipid-lowering effect is better than single fish oil, and is comparable to chemical drugs.The present application first realizes polypeptide-fish oil synergistic effect, provides safe and efficient new scheme for hyperlipidemia prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the technical field of cross-disciplinary research on bioactive peptides and functional oils, specifically relating to a lipid-lowering fish oil polypeptide composition and its applications. This composition is suitable for the prevention and treatment of hyperlipidemia and related cardiovascular diseases, and can be formulated into soft capsules, tablets, and other dosage forms. Background Technology

[0002] Hyperlipidemia is a major contributing factor to atherosclerosis and cardiovascular and cerebrovascular diseases, with a global prevalence exceeding 30%. Currently, mainstream lipid-lowering drugs such as statins (simvastatin) carry the risk of rhabdomyolysis (incidence 1–5%), while cholesterol absorption inhibitors (ezetimibe) and PCSK9 inhibitors (alixenobactam) suffer from high costs and the need for injection. Orlistat, as a lipase inhibitor, may cause fat-soluble vitamin deficiencies and gastrointestinal adverse reactions. Therefore, the development of safe, multi-target natural lipid-lowering components is urgently needed.

[0003] Fish oil is rich in omega-3 fatty acids (EPA / DHA), which have been shown to lower triglycerides, but its effect on regulating total cholesterol and LDL-C is limited when used alone. Although bioactive peptides have lipid-lowering potential, current research mostly focuses on single peptides, lacking synergistic systems with fish oil, and bifunctional peptides that simultaneously inhibit cholesterol esterase and lipase have not yet been discovered.

[0004] Currently, there are no reports of combining pumpkin seed peptides with fish oil for lipid-lowering effects. Traditional fish oil soft capsules do not incorporate active peptide components, resulting in limited functionality. This invention is the first to isolate the bifunctional peptide C2-1 (KRGGRFL) from pumpkin seed protein hydrolysate and form a synergistic lipid-lowering system by combining it with fish oil. This composition overcomes the limitations of single-component formulations, achieving multi-target regulation of four blood lipid indicators, filling a gap in existing technologies. Summary of the Invention

[0005] To address the aforementioned issues, this invention first provides a fish oil composition containing pumpkin seed-derived lipid-lowering active peptide C2-1, along with its preparation method and application.

[0006] In some embodiments, the composition comprises refined fish oil and pumpkin seed-derived lipid-lowering active peptide C2-1, said active peptide comprising 1–5% of the total mass of the composition, and said active peptide containing the amino acid sequence KRGGRFL;

[0007] This invention also provides a method for preparing a lipid-lowering fish oil polypeptide composition;

[0008] In some embodiments, the lyophilized active peptide C2-1 powder is mixed with refined fish oil in a certain proportion and homogenized by high-speed shearing at 10,000 rpm for 30 minutes under nitrogen protection to form a peptide-oil dispersion.

[0009] In some embodiments, the active peptide C2-1 is obtained through the following steps:

[0010] a) Pumpkin seed defatted protein was hydrolyzed by a combination of trypsin and alkaline protease;

[0011] b) Ultrafiltration separation of components with molecular weight <3kDa from enzymatic hydrolysate;

[0012] c) The active peak component C2-1 was separated and identified by DEAE anion exchange chromatography.

[0013] This invention also provides a lipid-lowering active peptide, the amino acid sequence of which contains KRGGRFL. This invention further provides the use of the lipid-lowering active peptide in the preparation of a medicament for treating or preventing hyperlipidemia.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] The active peptide C2-1 (KRGGRFL) provided by this invention simultaneously inhibits cholesterol esterase and lipase, breaking through the limitations of existing single-function peptides; compared with single fish oil, the composition further enhances the reduction of serum LDL-C and increases the rate of HDL-C increase in rats; at the same time, the overall lipid-lowering efficacy is superior to aliximab, and avoids the muscle toxicity of statins and the gastrointestinal side effects of orlistat. Attached Figure Description

[0016] Figure 1 Anion exchange chromatography elution profile of the <3kDa fraction of pumpkin seed protein hydrolysate.

[0017] Figure 2 Comparison of the in vitro inhibition rates of active peptides on cholesterol esterase (CE) and lipase (PL).

[0018] Figure 3 Changes in serum total cholesterol (TC) levels in rats.

[0019] Figure 4 Changes in serum triglyceride (TG) levels in rats.

[0020] Figure 5 Changes in low-density lipoprotein cholesterol (LDL-C) levels in rats.

[0021] Figure 6 Changes in high-density lipoprotein cholesterol (HDL-C) levels in rats. Detailed Implementation

[0022] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0023] Example 1: Preparation and isolation of lipid-lowering active peptides from pumpkin seeds

[0024] Dried pumpkin seeds (Cucurbita moschata Duch.) were mechanically dehulled, and then the kernels were pulverized to a particle size of approximately 60-80 mesh using a pulverizer to obtain pumpkin seed powder. The pumpkin seed powder was mixed with n-hexane at a material-to-liquid ratio of 1:5 (w / v), and defatted by stirring at room temperature (25±2℃) for 4 hours. This process was repeated twice to thoroughly remove fat-soluble impurities. The defatted pumpkin seed powder was then air-dried until no solvent residue remained. 100g of defatted pumpkin seed powder was added to 1000mL of deionized water (material-to-liquid ratio 1:10 w / v), and the pH was adjusted to 9.0±0.1 with 1M NaOH solution. Extraction was carried out by continuous stirring in a 50℃ water bath for 2 hours. The extract was centrifuged at 4℃ and 8000×g for 20 minutes, and the supernatant was collected. The pH of the supernatant was slowly adjusted to 4.5±0.1 (near the isoelectric point of pumpkin seed protein) with 1M HCl solution, and the mixture was allowed to stand for 1 hour to allow the protein to precipitate completely. Centrifuge again at 4℃ and 8000×g for 20 minutes, and collect the precipitate. Wash the precipitate twice with a small amount of deionized water to remove residual acid and some impurities. Redissolve the washed precipitate in an appropriate amount of deionized water and adjust the pH to 7.0 with 1M NaOH solution. Place the solution into a dialysis bag with a molecular weight cutoff of 3.5kDa and dialyze against flowing deionized water at 4℃ for 24 hours (changing the water every 4 hours) to remove small molecule salts. Freeze-dry the dialyzed solution to obtain pumpkin seed crude protein powder (approximately 35g, yield approximately 35%), and store at -20℃ for later use.

[0025] Weigh 10g of the prepared pumpkin seed crude protein powder and dissolve it in 500mL of phosphate buffer (0.05M, pH 8.0) (protein concentration approximately 2% w / v), stirring thoroughly until dissolved. Preheat the protein solution to the optimal temperature for enzymatic hydrolysis, 45℃. Use trypsin (YEASEN, catalog number: 40101ES25) and alkaline protease (LABLEAD, catalog number: A1412) for combined enzymatic hydrolysis. Weigh 0.2g each of trypsin and alkaline protease at 2% (w / w) of the substrate protein mass. Add both enzymes to the preheated protein solution and immediately place the solution on a thermostatic magnetic stirrer. Stir continuously for 4 hours at 45℃ and pH 8.0 (maintained constant using 1M NaOH). After hydrolysis, quickly place the hydrolysate in a boiling water bath for 10 minutes to inactivate the enzymes. Cool the enzyme-inactivated hydrolysate to room temperature (25±2℃), centrifuge at 4℃ and 10000×g for 20 minutes, and collect the supernatant, which is the pumpkin seed protein hydrolysate.

[0026] Prepare an ultrafiltration system (Millipore Amicon Ultra-0.5ml 3KD ultrafiltration tube UFC5003BK); perform ultrafiltration on the supernatant of the obtained pumpkin seed protein hydrolysate at an operating pressure of 0.4MPa and room temperature (25±2℃). Collect the permeate that successfully passes through the 3kDa ultrafiltration membrane, which is the peptide fraction with a molecular weight less than 3kDa (<3kDa peptide fraction). Freeze-dry the <3kDa peptide fraction solution to obtain a lyophilized powder (approximately 4.8g), store at -20℃, and use for subsequent fine separation.

[0027] A DEAE Sepharose FastFlow anion exchange chromatography column was used. The column was equilibrated with 5 column volumes (CV) of equilibration buffer A (20 mM Tris-HCl, pH 8.0) at a flow rate of 2.0 mL / min until the UV absorbance (280 nm) and conductivity baseline stabilized. The lyophilized <3 kDa peptide fraction was dissolved in buffer A (approximately 50 mg / mL), filtered through a 0.22 μm filter, and 10 mL (approximately 500 mg) was loaded onto the equilibrated column. The column was washed with buffer A for 3 CVs at a flow rate of 2.0 mL / min, and the unbound fraction was collected. A linear gradient elution was used. Elution buffer B was buffer A (20 mM Tris-HCl, pH 8.0, 1 M NaCl) containing 1 M NaCl. Throughout the process, UV absorption was monitored. Based on the UV absorption spectrum, different elution peak components were collected manually or automatically. The collected ion exchange peak components were then desalted and concentrated. The concentrate was then freeze-dried to obtain freeze-dried powders of each ion exchange component. (See attached image) Figure 1 .

[0028] Figure 1 The results showed that C1 and C2 elution peaks appeared in the UV absorption spectrum, which were used for further identification by subsequent mass spectrometry analysis.

[0029] Example 2: Mass spectrometry analysis and identification of bioactive peptides from ion-exchange chromatographic components

[0030] The lyophilized powders of anion exchange chromatography-separated components C1 and C2 were reconstituted with 0.1% formic acid aqueous solution (mass spectrometry grade) and then used... Desalting was performed using a C18 microcolumn, followed by elution with acetonitrile / water (70:30, containing 0.1% formic acid) and concentration. The final product was obtained using nano-liquid chromatography-tandem mass spectrometry (column: Acclaim). TMPepMap RSLC C18, 75 μm × 15 cm; mobile phase A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution), run at 300 mL / min for 60 min gradient elution (5-35% B linearly increasing). Mass spectrometry detection was performed in positive ion mode, with the primary scan range set to *m / z*300-1500 (resolution 120,000), and the secondary fragmentation selecting the top 20 strongest precursor ions (collision energy 28%, resolution 30,000). Targeted screening of +2 / +3 charged ions was conducted to capture characteristic signals of short peptides. Peptide Ranker performed preliminary screening of the above peptides, identifying peptides with a predicted bioactivity score >0.6. The identification results are shown in Table 1.

[0031] Table 1. Major short sequences identified

[0032] Components peptide molecular weight Charge state Confidence Peptide Ranker C1-1 LPYAQAK 0.89kDa +2 / +3 99.8％ 0.252009 C1-2 VSIPERY 0.86kDa +2 / +3 99.5％ 0.283453 C1-3 AGHPKYL 0.78kDa +2 / +3 99.9％ 0.661265 C1-4 TRQPVFK 0.88kDa +2 / +3 99.6％ 0.274309 C1-5 KPRYFLA 0.89kDa +2 / +3 99.4％ 0.619658

[0033] Components peptide molecular weight Charge state Confidence Peptide Ranker C2-1 KRGGRFL 0.83kDa +2 / +3 99.4％ 0.743563 C2-2 IRVKRWI 0.97kDa +2 / +3 99.2％ 0.282217 C2-3 HRFKVLR 0.96kDa +2 / +3 99.8％ 0.464168 C2-4 VQRKRVL 0.90kDa +2 / +3 99.3％ 0.103304 C2-5 AWGPRNV 0.80kDa +2 / +3 99.6％ 0.606177

[0034] Table 1 shows that the Peptide Ranker bioactivity prediction scores of C1-3, C1-5, C2-1, and C2-5 are >0.6. All of the above bioactive short peptides were synthesized by Nanjing Jietai Biotechnology Co., Ltd. (Nanjing, Jiangsu, China) with a purity greater than 98%, and were used for subsequent bioactivity function verification.

[0035] Example 3: Screening for lipid-lowering active peptides with good inhibitory properties using cholesterol esterase and lipase activity inhibition tests.

[0036] Dissolve the lipid-lowering active peptide samples (C1-3, C1-5, C2-1, C2-5) and the positive control (simvastatin) in an appropriate amount of buffer to prepare a stock solution of 1 mg / mL. Mix a certain amount of the stock solution with the cholesterol esterase solution in the reaction system and incubate at 37°C for 10 minutes. Then add the chromogenic substrate 4-nitrophenyl butyrate (4-NPPB) and continue incubation at 37°C for 30 minutes. Immediately after the reaction, measure the absorbance (A) of each reaction system at 405 nm using a microplate reader. Each sample and control was tested in triplicate. The cholesterol esterase inhibition rate was calculated using the following formula:

[0037] Cholesterol esterase inhibition rate (%) = [(Solvent A control group - Sample A group) / Solvent A control group] × 100%; where Solvent A control group is the absorbance value of the control reaction containing only buffer and enzyme (without sample), and Sample A group is the absorbance value of the reaction containing the active peptide to be tested. Results are shown in […]. Figure 2 .

[0038] Dissolve the lipid-lowering active peptide samples (C1-3, C1-5, C2-1, C2-5) and the positive control (orlistat) in an appropriate amount of buffer to prepare a stock solution of 1 mg / mL. Mix a certain amount of the stock solution with the lipase solution in the reaction system and incubate at 37°C for 10 minutes. Then add the chromogenic substrate 4-nitrophenyl myristate (4-NPP) and continue incubation at 37°C for 30 minutes. Immediately after the reaction, measure the absorbance (A) of each reaction system at 405 nm using a microplate reader. Each sample and control was tested in triplicate. The lipase inhibition rate was calculated using the following formula:

[0039] Lipase inhibition rate (%) = [(Solvent A control group - Sample A group) / Solvent A control group] × 100%; where Solvent A control group is the absorbance value of the control reaction containing only buffer and enzyme (without sample), and Sample A group is the absorbance value of the reaction containing the active peptide to be tested. Results are shown in […]. Figure 2 .

[0040] Figure 2 The results showed that at a concentration of 1 mg / mL, the C2-1 active peptide had a higher inhibition rate on cholesterol esterase or lipase activity than other active peptides; that is, the C2-1 active peptide had the best lipid-lowering activity, and it could not only inhibit cholesterol esterase, but also effectively inhibit lipase activity, and the inhibitory effect was comparable to that of simvastatin and orlistat.

[0041] Example 4: Verification of the lipid-lowering efficacy of lipid-lowering active peptide C2-1 in animals

[0042] Preparation of ordinary fish oil soft capsules: Deep-sea fish oil raw materials (EPA+DHA content ≥85%, acid value ≤1.0mg KOH / g) were taken, and free fatty acids were removed by molecular distillation (temperature 80℃, pressure 0.1Pa). The oil was filtered through a 0.45μm filter membrane to remove impurities, and 100g of refined fish oil was obtained. 0.1% (w / w) tocopherol was added as an antioxidant and stirred in the dark for 20min. Gelatin (Bloom strength 180±5) and glycerol were mixed in a ratio of 6:1 (w / w), and purified water was added to swell to 40% (w / w) water content. The mixture was then dissolved in a 50℃ water bath for 30min. A rotary capsule press was used, with the die temperature set at 40℃±1℃ and the injection rate at 5mL / min. Each capsule was filled with 100±5mg of fish oil.

[0043] Preparation of fish oil soft capsules containing lipid-lowering active peptide C2-1:

[0044] Deep-sea fish oil raw material (EPA+DHA content ≥85%, acid value ≤1.0mg KOH / g) was used to remove free fatty acids by molecular distillation (temperature 80℃, pressure 0.1Pa), and impurities were removed by filtration through a 0.45μm filter membrane to obtain 98g of refined fish oil. Then, the refined fish oil was emulsified and homogenized (10,000rpm, 5min) using gum arabic-maltodextrin composite wall material to form a stable emulsion, followed by spray drying (inlet air 180℃ / outlet air 80℃) to obtain fish oil microcapsules. 2g of C2-1 lyophilized peptide powder (purity ≥95%, particle size ≤50μm) was weighed, and then the peptide powder was combined with chitosan and alginic acid. Sodium was used as the wall material, and polypeptide microcapsules were prepared by ion cross-linking (1.5% CaCl2) and freeze drying. Fish oil microcapsules and polypeptide microcapsules were mixed and 0.1% tocopherol was added for antioxidant effect. Finally, they were encapsulated with gelatin-glycerol (6:1) soft capsule shell (mold temperature 40℃, injection rate 5mL / min) to make capsules containing 100±5mg of composite microcapsules per capsule. HPLC detection ensured that the C2-1 peptide retention rate was ≥98.5%. Stepwise microencapsulation technology was used to solve the oil-water compatibility problem and improve the stability of active ingredients.

[0045] Sixty healthy SPF-grade SD rats (male, 200±20g) were randomly divided into 5 groups (n=15): The blank control group was fed a normal maintenance diet plus saline by gavage (10mL / kg daily); the model group was fed a high-fat diet (formula: 78.8% basal diet + 10% lard + 10% egg yolk powder + 1% cholesterol + 0.2% bile salts) plus saline by gavage (10mL / kg daily); experimental group 1 was fed a high-fat diet plus ordinary fish oil soft capsules (300mg / kg daily by gavage, without active peptides); experimental group 2 was fed a high-fat diet plus fish oil soft capsules containing C2-1 active peptides (300mg / kg daily by gavage, containing C2-1 active peptides); and the positive control group was fed a high-fat diet plus alimab (trade name: Praluent) (2mg / mL daily injection).

[0046] After 6 weeks of continuous feeding, modeling was considered successful when serum TC > 6.2 mmol / L and TG > 1.8 mmol / L. Following successful modeling, the model group was administered the medication via gavage for 6 weeks. After the last administration, the patient was kept NPO for 12 hours, and venous blood was collected under anesthesia. Serum was separated by centrifugation at 3000 rpm for 15 minutes. Serum TC, TG, LDL-C, and HDL-C were measured using a Hitachi 3100 fully automated biochemical analyzer. Results are shown below. Figure 3-6 .

[0047] Figure 3-6The results showed that the four indicators of TC, TG, LDL-C, and HDL-C in the model group were significantly different from those in the blank control group, indicating that the hyperlipidemia model was successfully established. The experimental group 2 had a further reduction in TC, TG, and LDL-C levels compared to the experimental group 1, proving that the C2-1 peptide significantly enhanced the lipid-lowering effect of fish oil. In addition, the lipid-lowering active peptide C2-1 significantly increased the protective lipid (HDL-C) while reducing atherosclerotic lipids (LDL-C, TG, TC) by simultaneously inhibiting cholesterol esterase and lipase. Its comprehensive regulatory efficacy was superior to that of the single-target drug alimab.

[0048] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipid-lowering fish oil polypeptide composition, characterized in that: It is composed of refined fish oil and pumpkin seed-derived lipid-lowering active peptide C2-1, wherein the active peptide accounts for 1–5% of the total mass of the composition, and the amino acid sequence of the active peptide is KRGGRFL.

2. A lipid-lowering active peptide, characterized in that, The amino acid sequence of the active peptide is KRGGRFL.

3. The use of the composition according to claim 1 or the lipid-lowering active peptide according to claim 2 in the preparation of a medicament for treating or preventing hyperlipidemia.

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