Fish oil polypeptide composition capable of reducing blood fat and application of fish oil polypeptide composition

Abstract

The invention relates to a blood fat reducing fish oil polypeptide composition and application thereof, and belongs to the field of biological medicine and functional food. The composition comprises fish oil and pumpkin seed active peptide C2-1 (the molecular weight is less than 3kDa), wherein the active peptide accounts for 1-5% of the total weight of the composition. The preparation method comprises the following steps: performing composite enzymolysis on pumpkin seed protein, performing ultrafiltration separation, performing anion exchange chromatography purification to obtain C2-1 active peptide (KRGGRFL), and homogenizing the KRGGRFL and fish oil to obtain the stable dispersion. The composition significantly reduces serum total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) by synchronously inhibiting cholesterol esterase and lipase activity, and improves high density lipoprotein cholesterol (HDL-C). Animal experiments show that the lipid-lowering effect of the fish oil is superior to that of single fish oil and is equivalent to that of chemical drugs. The polypeptide-fish oil synergistic interaction is realized for the first time, and a safe and efficient new scheme is provided for hyperlipidemia prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioactive peptides and functional oils, and specifically relates to a hypolipidemic fish oil polypeptide composition and application thereof. The composition is suitable for preventing and treating hyperlipidemia and related cardiovascular diseases, and can be prepared into dosage forms such as soft capsules and tablets. BACKGROUND

[0002] Hyperlipidemia is the main inducement of atherosclerosis and cardiovascular and cerebrovascular diseases, and the global prevalence rate is over 30%. The mainstream lipid-lowering drugs such as statins (simvastatin) have the risk of rhabdomyolysis (incidence rate of 1-5%), and cholesterol absorption inhibitors (ezetimibe) and PCSK9 inhibitors (alirocumab) have the problems of high cost and injection administration. Orlistat as a lipase inhibitor may cause fat-soluble vitamin deficiency and digestive tract adverse reactions. Therefore, it is urgent to develop safe and multi-target natural lipid-lowering ingredients.

[0003] Fish oil is rich in omega-3 fatty acids (EPA / DHA) and has been proved to reduce triglycerides, but the regulation effect on total cholesterol and LDL-C is limited when used alone. Although active polypeptides have the potential to lower lipid, existing researches mainly focus on single peptide segments, lack of synergistic system with fish oil, and no bifunctional peptide that simultaneously inhibits cholesterol esterase and lipase has been found.

[0004] There is no report on the combination of pumpkin seed peptide and fish oil for lowering lipid, and traditional fish oil soft capsules do not introduce active peptide components, resulting in single function. The present application first isolates a bifunctional peptide C2-1 (KRGGRFL) from pumpkin seed protein hydrolysate, and forms a synergistic lipid-lowering system by compounding with fish oil. The composition breaks through the limitation of single component, realizes multi-target regulation of four indicators of blood lipid, and fills the gap in the prior art. SUMMARY

[0005] To solve the above problems, the present application first provides a fish oil composition containing hypolipidemic active peptide C2-1 from pumpkin seeds and a preparation method and application thereof.

[0006] In some embodiments, the composition comprises refined fish oil and hypolipidemic active peptide C2-1 from pumpkin seeds, the active peptide accounts for 1-5% of the total mass of the composition, and the amino acid sequence of the active peptide comprises KRGGRFL;

[0007] The present application also provides a preparation method of the hypolipidemic fish oil polypeptide composition.

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

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

[0010] a) pumpkin seed defatted protein is subjected to trypsin and alkaline protease complex enzymolysis;

[0011] b) the enzymolysis solution is subjected to ultrafiltration to separate components with a molecular weight of <3 kDa;

[0012] c) the components C2-1 in the active peak are separated and identified by DEAE anion exchange chromatography.

[0013] The present application also provides a blood lipid-lowering active peptide, and the amino acid sequence of the active peptide comprises KRGGRFL. The present application also provides a use of a blood lipid-lowering active peptide in the preparation of a medicament for treating or preventing hyperlipidemia.

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

[0015] The active peptide C2-1 (KRGGRFL) provided by the present application simultaneously inhibits cholesterol esterase and lipase, breaking through the limitations of existing single-function peptides; compared with single fish oil, the composition additionally increases the reduction amplitude of rat serum LDL-C and increases the HDL-C increase rate; at the same time, the comprehensive lipid-lowering efficiency is better than that of alirocumab, and muscle toxicity of statins and digestive tract side effects of orlistat are avoided. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Anion exchange chromatography elution spectrum of <3 kDa components of pumpkin seed protein enzymolysis products.

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

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

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

[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 DESCRIPTION

[0022] To make the technical problems, technical solutions and advantages of the present application clearer, specific embodiments will be described in detail below with reference to the drawings.

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

[0024] Take dried pumpkin seeds (Cucurbita moschata Duch.), after mechanical shelling, use a pulverizer to crush the kernel to a particle size of about 60-80 mesh to obtain pumpkin seed powder; mix the pumpkin seed powder with n-hexane at a material-liquid ratio of 1:5 (w / v), and stir to defat at room temperature (25±2°C) for 4 hours, repeat the operation 2 times to fully remove fat-soluble impurities. The defatted pumpkin seed powder is dried by ventilation until there is no solvent residue. Take 100 g of defatted pumpkin seed powder, add 1000 mL of deionized water (material-liquid ratio 1:10 w / v), and adjust the pH to 9.0±0.1 with 1M NaOH solution. Stir continuously in a 50°C water bath for 2 hours. Centrifuge the extract at 4°C, 8000xg for 20 minutes, and collect the supernatant. Slowly adjust the pH of the supernatant to 4.5±0.1 (near the isoelectric point of pumpkin seed protein) with 1M HCl solution, and let stand for 1 hour to allow the protein to fully precipitate. Centrifuge again at 4°C, 8000xg for 20 minutes, and collect the precipitate. Wash the precipitate with a small amount of deionized water twice to remove residual acid solution and part of the impurities. Redissolve the washed precipitate in an appropriate amount of deionized water, and adjust the pH to 7.0 with 1M NaOH solution. The solution is loaded into a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed against flowing deionized water at 4°C for 24 hours (change water every 4 hours) to remove small molecule salts. Freeze-dry the dialyzed solution to obtain pumpkin seed crude protein powder (about 35 g, yield about 35%), and store at -20°C for later use.

[0025] Take 10 g of the pumpkin seed crude protein powder prepared above, dissolve in 500 mL of phosphate buffer (0.05M, pH 8.0) (protein concentration about 2% w / v), and stir to dissolve. Preheat the protein solution to the optimal temperature for enzyme hydrolysis, 45°C; select trypsin (YEASEN, Catalog No.: 40101ES25) and alkaline protease (LABLEAD, Catalog No.: A1412) for complex enzyme hydrolysis. Take 2% (w / w) of trypsin and alkaline protease (0.2 g each) based on the mass of substrate protein; add the two enzymes to the preheated protein solution, immediately place it in a constant temperature magnetic stirrer, and continuously stir at 45°C, pH 8.0 (use 1M NaOH to maintain constant) for 4 hours. After enzyme hydrolysis, immediately place the enzyme hydrolysate in a boiling water bath for 10 minutes to inactivate the enzyme activity. Cool the enzyme-inactivated enzyme hydrolysate to room temperature (25±2°C), centrifuge at 4°C, 10000xg for 20 minutes, and collect the supernatant, which is the pumpkin seed protein hydrolysate.

[0026] Ultrafiltration system was prepared (Millipore Amicon Ultra-0.5ml 3KD ultrafiltration tube UFC5003BK); the obtained pumpkin seed protein hydrolysate supernatant was subjected to ultrafiltration under the conditions of operating pressure 0.4 MPa and room temperature (25±2℃). The filtrate (Permeate) that smoothly passed through the 3kDa ultrafiltration membrane was collected, which was the peptide component with molecular weight less than 3kDa (<3kDa peptide component). The <3kDa peptide component solution was freeze-dried to obtain a freeze-dried powder (about 4.8g), which was stored at -20℃ and used for subsequent fine separation.

[0027] DEAE Sepharose FastFlow anion exchange chromatography column was selected; the chromatography column was equilibrated with 5 column volumes (CV) of equilibration buffer A (20mM Tris-HCl, pH 8.0) at a flow rate of 2.0mL / min until the ultraviolet absorption (280nm) and conductivity baseline were stable. The obtained <3kDa peptide component freeze-dried powder was dissolved with buffer A (concentration about 50mg / mL), filtered through a 0.22μm filter membrane, and then 10mL (about 500mg) was loaded into the equilibrated chromatography column. 3CV of buffer A was used for elution at a flow rate of 2.0mL / min, and the unbound component was collected; linear gradient elution was used. Elution buffer B was buffer A (20mM Tris-HCl, pH 8.0, 1M NaCl) containing 1M NaCl. The ultraviolet absorption was monitored throughout the process, and different elution peak components were collected manually or automatically according to the ultraviolet absorption spectrum; the collected ion exchange peak components were desalted and concentrated, and then the concentrated solution was freeze-dried to obtain ion exchange component freeze-dried powder, as shown in Table 1. Figure 1 .

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

[0029] Example 2: Mass spectrometry analysis of ion exchange chromatography components and identification of active peptides

[0030] The freeze-dried powder of anion exchange chromatography separation components C1 and C2 was reconstituted with mass spectrometry grade 0.1% formic acid aqueous solution, and then desalted using C18 microcolumn, eluted with acetonitrile / water (70:30 containing 0.1% formic acid) and concentrated. C18 microcolumn desalting, acetonitrile / water (70:30 containing 0.1% formic acid) elution and concentration. Nanoliter liquid chromatography-tandem mass spectrometry system (chromatography column: Acclaim Pep TMPepMap RSLC C18, 75 pm x 15 cm; mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile, running a 60 min gradient (5-35% B linear increase) at a flow rate of 300 mL / min. Mass spectrometry detection was performed in positive ion mode with a first scan range *m / z* 300-1500 (resolution 120,000), second fragmentation selecting the top 20 precursor ions (collision energy 28%, resolution 30,000), and targeted screening of +2 / +3 charged ions to capture short peptide signatures. Peptide Ranker was used to screen the above polypeptides, and peptides with a bioactivity prediction score >0.6 were selected. The identification results are shown in Table 1.

[0031] Table 1: Main short sequences identified

[0032] Component Peptide Segment Molecular Weight Charge State Confidence Peptide Ranker C1-1 LPYAQAK 0.89 kDa +2 / +3 99.8％ 0.252009 C1-2 VSIPERY 0.86 kDa +2 / +3 99.5％ 0.283453 C1-3 AGHPKYL 0.78 kDa +2 / +3 99.9％ 0.661265 C1-4 TRQPVFK 0.88 kDa +2 / +3 99.6％ 0.274309 C1-5 KPRYFLA 0.89 kDa +2 / +3 99.4％ 0.619658

[0033] Component Peptide Segment Molecular Weight Charge State Confidence Peptide Ranker C2-1 KRGGRFL 0.83 kDa +2 / +3 99.4％ 0.743563 C2-2 IRVKRWI 0.97 kDa +2 / +3 99.2％ 0.282217 C2-3 HRFKVLR 0.96 kDa +2 / +3 99.8％ 0.464168 C2-4 VQRKRVL 0.90 kDa +2 / +3 99.3％ 0.103304 C2-5 AWGPRNV 0.80 kDa +2 / +3 99.6％ 0.606177

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

[0035] Example 3: Cholesterol esterase activity inhibition and lipase enzyme activity inhibition test to screen lipid-lowering active peptides with good inhibition performance

[0036] The lipid-lowering active peptide samples (C1-3, C1-5, C2-1, and C2-5) to be tested and the positive control (simvastatin) were dissolved with an appropriate amount of buffer to prepare a 1 mg / mL stock solution. A certain amount of the stock solution was mixed with the cholesterol esterase solution in the reaction system, and incubated at 37°C for 10 minutes. Then, the color developing substrate 4-nitrophenyl butyrate (4-NPPB) was added, and the reaction was continued at 37°C for 30 minutes. After the reaction was completed, the absorbance value (A) of each reaction system was immediately determined at 405 nm using an enzyme label instrument. Each sample and control was set in triplicate. The cholesterol esterase inhibition rate was calculated according to the following formula:

[0037] Cholesterol esterase inhibition rate (%) = [(A solvent control group - A sample group) / A solvent control group] x 100%; wherein, A solvent control group is the absorbance value of the control reaction containing only the buffer and the enzyme (without the sample), and A sample group is the absorbance value of the reaction containing the active peptide sample to be tested. The results are shown in Table 2. Figure 2 .

[0038] The sample (C1-3, C1-5, C2-1, C2-5) and positive control (orlistat) were dissolved in buffer to prepare a stock solution of 1 mg / mL. A certain amount of the stock solution was mixed with lipase solution in the reaction system, and incubated at 37°C for 10 minutes. Then, the color developing substrate 4-nitrophenyl myristate (4-NPP) was added, and the reaction was continued at 37°C for 30 minutes. After the reaction, the absorbance value (A) of each reaction system was measured at 405 nm using an enzyme label instrument. Each sample and control was set in triplicate. The lipase inhibition rate was calculated according to the following formula:

[0039] Lipase inhibition rate (%) = [(A solvent control - A sample group) / A solvent control] x 100%; wherein, A solvent control is the absorbance value of the control reaction containing only buffer and enzyme (without sample), and A sample group is the absorbance value of the reaction containing the sample to be tested. The results are shown in Table 2. Figure 2 .

[0040] Figure 2 The results show that at a concentration of 1 mg / mL, the C2-1 active peptide has a higher inhibition rate on cholesterol esterase activity or lipase enzyme activity than other active peptides. That is, the C2-1 active peptide has the best lipid-lowering activity, which can not only inhibit cholesterol esterase, but also effectively inhibit lipase activity, and the inhibition activity is comparable to that of simvastatin and orlistat.

[0041] Example 4: Animal lipid-lowering efficacy verification of the lipid-lowering active peptide C2-1

[0042] Preparation of ordinary fish oil soft capsules: Take deep sea fish oil raw material (EPA+DHA content ≥85%, acid value ≤1.0 mg KOH / g), remove free fatty acids by molecular distillation (temperature 80°C, pressure 0.1 Pa), and remove impurities by passing through a 0.45 μm filter to obtain 100 g of refined fish oil; add 0.1% (w / w) tocopherol as an antioxidant, stir in the dark for 20 min; mix gelatin (Bloom strength 180±5) and glycerol at a ratio of 6:1 (w / w), swell with purified water to 40% (w / w) moisture, and dissolve the gel at 50°C for 30 min; use a rotary capsule filling machine, set the mold temperature to 40°C±1°C, and fill each capsule with 100±5 mg of fish oil.

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

[0044] Take the raw material of deep sea fish oil (EPA+DHA content ≥ 85%, acid value ≤ 1.0 mg KOH / g), remove free fatty acids by molecular distillation (temperature 80℃, pressure 0.1 Pa), and remove impurities by 0.45μm filter membrane to obtain 98g of refined fish oil; then the refined fish oil is emulsified and homogenized (10,000 rpm, 5 min) with arabic gum-malt dextrin composite wall material to form a stable emulsion, and then spray dried (inlet temperature 180℃ / outlet temperature 80℃) to obtain fish oil microcapsules; take 2g of C2-1 freeze-dried peptide powder (purity ≥ 95%, particle size ≤ 50μm), and then prepare peptide microcapsules by ion cross-linking (1.5% CaCl2) and freeze-drying with chitosan and sodium alginate as wall material; mix the fish oil microcapsules and peptide microcapsules, add 0.1% tocopherol antioxidant, and finally coat with gelatin-glycerol (6:1) soft capsule shell (mold temperature 40℃, injection rate 5mL / min) to prepare capsules containing 100±5mg of composite microcapsules per capsule. HPLC detection ensures that the retention rate of C2-1 peptide is ≥98.5%, and the stepwise microencapsulation technology solves the problem of oil-water compatibility and improves the stability of active ingredients.

[0045] Select 60 healthy SPF SD rats (male, 200±20g), and randomly divide them into 5 groups (n=15): among them, the blank control group: ordinary maintenance feed + physiological saline gavage (10mL / kg daily gavage); model group: high-fat feed (formula: 78.8% basic feed + 10% lard + 10% egg yolk powder + 1% cholesterol + 0.2% cholate) + physiological saline gavage (10mL / kg daily gavage); experimental group 1: high-fat feed + ordinary fish oil soft capsules (300mg / kg daily gavage, without active peptide); experimental group 2: high-fat feed + fish oil soft capsules containing C2-1 active peptide (300mg / kg daily gavage, containing C2-1 active peptide); positive control group: high-fat feed + Alirocumab (trade name: Praluent) (2mg / mL daily injection);

[0046] Continue to feed for 6 weeks, and confirm that the model group has serum TC > 6.2mmol / L and TG > 1.8mmol / L, which means that the modeling is successful; after the modeling is successful, continue gavage for 6 weeks; after the last administration, fast for 12 hours, and collect venous blood under anesthesia, and centrifuge at 3000rpm for 15 minutes to separate serum; determine serum TC, TG, LDL-C, and HDL-C by Hitachi 3100 automatic biochemical analyzer, and the results are shown in Figures 3-6 .

[0047] Figures 3-6The results show that the four indicators of TC, TG, LDL-C and HDL-C of the model group have significant differences compared with the blank control group, indicating that the hyperlipidemia model is successfully established; the experimental group 2 reduces the contents of TC, TG and LDL-C more than the experimental group 1, proving that the C2-1 peptide significantly enhances the lipid-lowering effect of fish oil; in addition, the antihyperlipidemic active peptide C2-1 significantly improves the protective lipid (HDL-C) by simultaneously inhibiting cholesterol esterase and lipase, while reducing the atherosclerosis-causing lipid (LDL-C, TG, TC), and its comprehensive regulation efficiency is better than that of the single-target drug alirocumab.

[0048] The above is the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A lipid-lowering fish oil polypeptide composition, characterized in that: It contains 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 contains KRGGRFL.

2. A method for preparing the lipid-lowering fish oil polypeptide composition according to claim 1, characterized in that: The freeze-dried active peptide C2-1 powder was 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.

3. The method according to claim 2, characterized in that, The active peptide C2-1 is obtained through the following steps: a) Pumpkin seed defatted protein was hydrolyzed by a combination of trypsin and alkaline protease; b) Ultrafiltration separation of components with molecular weight <3kDa from enzymatic hydrolysate; c) The active peak component C2-1 was separated and identified by DEAE anion exchange chromatography.

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

5. The use of the composition according to claim 1 or the lipid-lowering active peptide according to claim 4 in the preparation of a medicament for treating or preventing hyperlipidemia.

Images