A lipid-lowering hexapeptide from large yellow croaker scales, its preparation method and application

Abstract

The present invention discloses a hypolipidemic hexapeptide derived from large yellow croaker scales, its preparation method and application. The present invention uses large yellow croaker scales as raw materials, through degreasing and decalcification treatment, ultrasonic combined enzymatic hydrolysis, membrane ultrafiltration, and chromatographic separation and purification to obtain the hypolipidemic hexapeptide Ser-His-Gly-Pro-Cys-Phe (SHGPCF), and the molecular weight measured by ESI-MS is 674.8 Da. The hypolipidemic peptide SHGPCF from large yellow croaker scales prepared by the present invention can significantly reduce the lipid accumulation in HepG2 cells induced by oleic acid (OA), and significantly reduce the contents of total cholesterol (TC) and triglyceride (TG) in the cell model, and has good hypolipidemic activity. Moreover, SHGPCF has no significant effect on the viability of HepG2 cells, and the activity of SHGPCF is significant and has no obvious toxicity, and can be applied to drugs and health products related to hypolipidemia.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a bighead croaker scale hypolipidemic hexapeptide, its preparation method and application. Background Art

[0002] Hyperlipidemia refers to the situation where the total cholesterol (TC) and / or triglyceride (TG) in the blood is too high or the high-density lipoprotein cholesterol (HDL-C) is too low, exceeding the reasonable range value, which is called dyslipidemia in modern medicine. Hyperlipidemia is a common disease in life. If not treated in time, it will induce related diseases such as coronary heart disease, arteriosclerosis, pancreatitis, etc., posing a threat to human health. However, with the increasing consumption of high-calorie foods and the decreasing daily activity level, the number of hyperlipidemia patients is increasing day by day and showing a trend of younger age. The drug treatment of hyperlipidemia has a quick effect, but hypolipidemic drugs have certain toxic and side effects. Therefore, taking health products supplemented with diet therapy is an important way to treat hyperlipidemia, which is of great significance for improving the quality of life of middle-aged and elderly people. Summary of the Invention

[0003] Based on this, the applicant uses bighead croaker scales as raw materials, and uses enzymatic hydrolysis technology and chromatographic preparation technology to prepare hypolipidemic peptides. This hypolipidemic peptide is safe and has no toxic and side effects, and can be used for the development of hypolipidemic related functional products. The present invention provides a bighead croaker scale hexapeptide with hypolipidemic efficacy, which can be used as a safe additive for drugs, health foods and foods.

[0004] A bighead croaker scale hexapeptide with hypolipidemic efficacy, its amino acid sequence is Ser-His-Gly-Pro-Cys-Phe (SHGPCF), and the molecular weight measured by ESI-MS is 674.8 Da.

[0005] A preparation method of a bighead croaker scale hexapeptide with hypolipidemic efficacy, which includes the following steps:

[0006] 1) Pretreatment of bighead croaker scales: Add bighead croaker scales to NaOH solution, soak at 4°C for 3 - 5 h to remove non-collagen proteins; the treated bighead croaker scales are repeatedly washed with distilled water until neutral, drained, added to EDTA-Na solution with pH 7.4, soaked at 4°C for 24 - 48 h (replace the EDTA solution 1 - 2 times a day), washed 3 times with distilled water, dried and pulverized;

[0007] 2) Enzymatic hydrolysis of large yellow croaker scales: Take the pre-treated large yellow croaker scales above, add distilled water, adjust the temperature to 35 - 40 °C, adjust the pH value to 1.5 - 2.0, perform ultrasonic treatment at 42 KHZ and 300 W for 20 - 30 min, add pepsin, hydrolyze for 3 - 4 h, inactivate the enzyme at 95 °C for 15 min; when the sample cools down to 45 - 50 °C, adjust the pH value to 7.0 - 8.5, add neutral protease, react for 2 - 3 h, inactivate the enzyme at 95 °C for 15 min, cool to room temperature, centrifuge at 9000 rmp for 15 min, and collect the supernatant, namely the enzymatic hydrolysate of large yellow croaker scales;

[0008] 3) Preparation of lipid-lowering oligopeptides from large yellow croaker scales: The enzymatic hydrolysate of large yellow croaker scales is fractionated by ultrafiltration membranes with a molecular weight cut-off of 1 kDa, 3 kDa, and 5 kDa, the fractionated components are collected, and the effects of each component on lipid accumulation in the HepG2 cell model are detected. The ultrafiltration fraction with the best lipid-lowering effect is purified by Sephadex LH-20 column chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) in sequence to obtain lipid-lowering oligopeptides from large yellow croaker scales.

[0009] In some embodiments of the present invention, the concentration of the NaOH solution in step 1) is 0.05 mol / L, and the weight-to-volume ratio of the large yellow croaker scales to the NaOH solution is 1 g:10 - 15 mL.

[0010] In some embodiments of the present invention, the concentration of the EDTA-Na solution in step 1) is 0.5 mol / L, and the weight-to-volume ratio of the large yellow croaker scales to the EDTA-Na solution is 1 g:6 - 10 mL.

[0011] In some embodiments of the present invention, the weight ratio of the large yellow croaker scales to distilled water in step 2) is 1:10.

[0012] In some embodiments of the present invention, the addition amount of pepsin in step 2) is 1.0 - 1.2% of the weight of the large yellow croaker scales.

[0013] In some embodiments of the present invention, the addition amount of neutral protease in step 2) is 1.0 - 1.5% of the weight of the large yellow croaker scales.

[0014] In some embodiments of the present invention, the specific processes of Sephadex LH-20 column chromatography and RP-HPLC purification in step 3) are as follows:

[0015] Gel column chromatography: The ultrafiltration fraction with the best lipid-lowering effect mentioned above was dissolved in double-distilled water to prepare a solution with a concentration of 25 - 30 mg / mL, and separated by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography (2.0×100 cm), eluted with double-distilled water at a flow rate of 0.5 - 0.6 mL / min. According to the gel chromatography chromatogram at 214 nm, each chromatographic peak fraction was collected, and the effect of each chromatographic peak fraction on lipid accumulation in the HepG2 cell model was measured. The chromatographic peak fraction with the best effect of reducing lipid accumulation was selected and freeze-dried to obtain the lipid-lowering gel chromatography hydrolysate of large yellow croaker scales.

[0016] RP-HPLC purification: The lipid-lowering gel chromatography hydrolysate of large yellow croaker scales mentioned above was dissolved in double-distilled water to prepare a solution with a concentration of 90 - 100 μg / mL, and purified by RP-HPLC. According to the lipid-lowering activity of the prepared polypeptide, 1 polypeptide with high lipid-lowering activity was obtained. Its sequence was determined as: Ser-His-Gly-Pro-Cys-Phe (SHGPCF), and the molecular weight was determined by ESI-MS to be 674.8 Da.

[0017] In some embodiments of the present invention, the RP-HPLC conditions are as follows: injection volume 150 - 200 μL; chromatographic column Hypersil 300A C18 (250 mm×10.0 mm, 10 μm); mobile phase: 60% acetonitrile; elution rate 1.5 - 2.0 mL / min; ultraviolet detection wavelength 214 nm.

[0018] On the other hand, the present invention provides the application of the above-mentioned lipid-lowering oligopeptide SHGPCF of large yellow croaker scales in drugs and / or health foods.

[0019] Compared with the prior art, the lipid-lowering oligopeptide SHGPCF of large yellow croaker scales provided by the present invention can significantly reduce the contents of triglyceride (TG) and total cholesterol (TC) in the lipid accumulation model of HepG2 cells at a concentration of 10 μM, showing good lipid-lowering activity. Ser-His-Gly-Pro-Cys-Phe (SHGPCF) has the advantages of being safe, non-toxic, and having strong lipid-lowering activity, and can be used as a drug and / or health food. Description of the Drawings

[0020] Figure 1 It is the effect of the ultrafiltration fraction of the enzymatic hydrolysate of large yellow croaker scales of the present invention on the lipid content in the lipid accumulation model of HepG2 cells at a concentration of 5 mg / mL.

[0021] Figure 2 It is the chromatogram of hydroxypropyl dextran gel (Sephadex LH-20) chromatography of the present invention.

[0022] Figure 3Effect of the enzymatic hydrolysate component prepared from Sephadex LH-20 on lipid content in the HepG2 cell lipid accumulation model at a concentration of 10 mg / mL.

[0023] Figure 4 RP-HPLC analysis of the enzymatic hydrolysate prepared from Sephadex LH-20.

[0024] Figure 5 Effect of the RP-HPLC separated component on lipid content in the HepG2 cell lipid accumulation model at a concentration of 5 mg / mL.

[0025] Figure 6 Mass spectrum of Ser-His-Gly-Pro-Cys-Phe (SHGPCF).

[0026] Figure 7 Structure of Ser-His-Gly-Pro-Cys-Phe (SHGPCF).

[0027] Figure 8 Effect of Ser-His-Gly-Pro-Cys-Phe (SHGPCF) on the content of triglyceride (TG) in the HepG2 cell lipid accumulation model.

[0028] Figure 9 Effect of Ser-His-Gly-Pro-Cys-Phe (SHGPCF) on the content of total cholesterol (TC) in the HepG2 cell lipid accumulation model.

[0029] Figure 10 Effect of Ser-His-Gly-Pro-Cys-Phe (SHGPCF) on the viability of HepG2 cells. Detailed implementation manners

[0030] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] Normal group: HepG2 cells that grow in the culture medium without any treatment.

[0032] Model group: HepG2 cells treated with a 1 mmol / L fatty acid (oleic acid: palmitic acid 2:1) solution for 24 h.

[0033] Positive control group: HepG2 cells treated with 10 μM simvastatin and a 1 mmol / L fatty acid (oleic acid: palmitic acid 2:1) solution for 24 h.

[0034] SHGPCF group: HepG2 cells treated with a solution containing 10 μM oligopeptide SHGPCF and 1 mmol / L fatty acids (oleic acid: palmitic acid = 2:1) for 24 h.

[0035] A hypolipidemic hexapeptide derived from large yellow croaker scales has the following preparation process: large yellow croaker scales → pretreatment → ultrasonic treatment and enzymatic hydrolysis → enzymatic hydrolysate → ultrafiltration → gel filtration chromatography → RP-HPLC separation and purification → hypolipidemic hexapeptide → functional evaluation.

[0036] The specific steps are as follows:

[0037] 1) Pretreatment of large yellow croaker scales: Add large yellow croaker scales to NaOH solution (0.05 mol / L) at a solid-liquid ratio of 1 g:12 mL and soak at 4 °C for 5 h to remove non-collagen proteins; after treatment, wash the large yellow croaker scales repeatedly with distilled water until neutral, drain, add EDTA-Na solution (0.5 mol / L) with pH 7.4 at a solid-liquid ratio of 1 g:8 mL, soak at 4 °C for 48 h (change the EDTA solution 1 - 2 times a day), wash with distilled water 3 times, dry and pulverize.

[0038] 2) Enzymatic hydrolysis of large yellow croaker scales: Take the pretreated large yellow croaker scales, add distilled water at a volume ratio of 1:10 (v / v), adjust the temperature to 37 °C, adjust the pH value to 1.5, perform ultrasonic treatment at 42 KHZ and 300 W for 25 min, add 1.2% pepsin based on the weight of the scales, hydrolyze for 4 h, inactivate the enzyme at 95 °C for 15 min; when the sample temperature drops to 48.5 °C, adjust the pH value to 7.5, add 1.2% neutral protease, react for 3 h, inactivate the enzyme at 95 °C for 15 min, cool to room temperature, centrifuge at 9000 rmp for 15 min, and collect the supernatant, which is the enzymatic hydrolysate of large yellow croaker scales (LSH).

[0039] 3) Preparation of hypolipidemic oligopeptides from large yellow croaker scales: The enzymatic hydrolysate of large yellow croaker scales (LSH) is fractionated by ultrafiltration membranes with a cut-off molecular weight of 1 kDa, 3 kDa, and 5 kDa, and the fractionated components LSH-1 (MW < 1 kDa), LSH-2 (1 kDa < MW < 3 kDa), LSH-3 (3 kDa < MW < 5 kDa), and LSH-4 (MW > 5 kDa) are collected. The effects of LSH1 - LSH4 on lipid accumulation in the HepG2 cell model are measured (see Figure 1 ), and LSH-1 has the best effect on reducing lipid accumulation. LSH-1 is purified successively by Sephadex LH-20 and reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain hypolipidemic oligopeptides from large yellow croaker scales. Their structures are determined using an amino acid sequence analyzer and mass spectrometry. The specific process is as follows:

[0040] ① Hydroxypropyl dextran gel (Sephadex LH-20) chromatography: Dissolve the above-mentioned LSH-1 in double-distilled water to prepare a solution with a concentration of 25 mg / mL. Separate it by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography (2.0×100 cm), elute with double-distilled water at a flow rate of 0.5 mL / min. According to the gel filtration chromatogram at 214 nm (see Figure 2 ), collect the components of each chromatographic peak (LSH-1A to LSH-1C), and determine the effect of each chromatographic peak component on lipid accumulation in the HepG2 cell model (see Figure 3 ). It is determined that LSH-1B has the best effect on reducing lipid accumulation, that is, the gel filtration enzymatic hydrolysate;

[0041] ② RP-HPLC purification: Dissolve the above-mentioned LSH-1B in double-distilled water to prepare a solution with a concentration of 95 μg / mL, and purify it by RP-HPLC (injection volume 200 μL; chromatographic column Hypersil 300A C18 (250 mm×10.0 mm, 10 μm); mobile phase: 50% acetonitrile; elution speed 2.0 mL / min; UV detection wavelength 214 nm). According to the absorbance curve at 214 nm (see Figure 4 ) and the lipid-lowering activity (see Figure 5 ), obtain 1 highly active lipid-lowering oligopeptide (LHP6);

[0042] ③ Structure detection: Collect LHP6 with the highest lipid-lowering activity. After detection by RP-HPLC and meeting the sequencing requirements, use a protein / peptide sequencer to determine the amino acid sequence as Ser-His-Gly-Pro-Cys-Phe (SHGPCF) (see Figure 6 ), and determine the molecular weight by ESI-MS to be 674.8 Da (see Figure 7 );

[0043] ④ Function evaluation: Refer to the literature [Cheng Jing, Liu Ying, Liu Yaojie, Zhao Jiang, Ji Yanglin, Liu Dong, Wang Hao. Carnosic acid reduces free fatty acid-induced lipid accumulation in HepG2 cells by activating AMPK [J]. Food Science, 2020, 41(11): 171-178] to establish a HepG2 cell lipid accumulation model, and evaluate the lipid-lowering effect of the lipid-lowering hexapeptide Ser-His-Gly-Pro-Cys-Phe (SHGPCF) from large yellow croaker scales. The results show that: at a concentration of 10 μM, SHGPCF can significantly reduce lipid accumulation in the cell model and significantly reduce the content of triglyceride (TG) (see Figure 8 ) and total cholesterol (TC) (see Figure 9 ). Therefore, Ser-His-Gly-Pro-Cys-Phe (SHGPCF) has significant lipid-lowering activity and has no significant effect on the viability of HepG2 cells.Figure 10 ).

[0044] Finally, it should be noted that the above is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiment, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A hexapeptide from Pseudosciaena crocea scales with lipid-lowering efficacy, characterized in that The amino acid sequence of the hexapeptide is Ser-His-Gly-Pro-Cys-Phe (SHGPCF).

2. The preparation method of the hexapeptide from Pseudosciaena crocea scales with lipid-lowering efficacy according to claim 1, characterized in that It includes the following steps: 1) Pretreatment of large yellow croaker scales: Add large yellow croaker scales to a NaOH solution, soak at 4°C for 3 - 5 h to remove non-collagen proteins; wash the treated large yellow croaker scales repeatedly with distilled water until neutral, drain, add to an EDTA-Na solution with a pH of 7.4, soak at 4°C for 24 - 48 h (replace the EDTA solution 1 - 2 times a day), wash 3 times with distilled water, dry and pulverize; 2) Enzymatic hydrolysis of large yellow croaker scales: Take the pretreated large yellow croaker scales obtained above, add distilled water, adjust the temperature to 35 - 40°C, adjust the pH value to 1.5 - 2.0, perform ultrasonic treatment at 42 KHZ and 300 W for 20 - 30 min, add pepsin, hydrolyze for 3 - 4 h, inactivate the enzyme at 95°C for 15 min; when the sample cools to 45 - 50°C, adjust the pH value to 7.0 - 8.5, add neutral protease, react for 2 - 3 h, inactivate the enzyme at 95°C for 15 min, cool to room temperature, centrifuge at 9000 rmp for 15 min, collect the supernatant, which is the enzymatic hydrolysate of large yellow croaker scales; 3) Preparation of lipid-lowering oligopeptides from large yellow croaker scales: Subject the enzymatic hydrolysate of large yellow croaker scales to ultrafiltration through ultrafiltration membranes with a molecular weight cut-off of 1 kDa, 3 kDa, and 5 kDa for fractionation, collect the fractionated components, and detect the effects of each component on lipid accumulation in the HepG2 cell model. The ultrafiltration fraction with the best lipid-lowering effect is purified successively by Sephadex LH-20 column chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain lipid-lowering oligopeptides from large yellow croaker scales; In step 1), the concentration of the NaOH solution is 0.05 mol / L, and the weight-to-volume ratio of the large yellow croaker scales to the NaOH solution is 1 g:10 - 15 mL; In step 1), the concentration of the EDTA-Na solution is 0.5 mol / L, and the weight-to-volume ratio of the large yellow croaker scales to the EDTA-Na solution is 1 g:6 - 10 mL; In step 2), the weight ratio of the large yellow croaker scales to distilled water is 1:10; in step 2), the addition amount of pepsin is 1.0 - 1.2% of the weight of the large yellow croaker scales; in step 2), the addition amount of neutral protease is 1.0 - 1.5% of the weight of the large yellow croaker scales; The Sephadex LH-20 column chromatography step in step 3) is as follows: Gel column chromatography: Dissolve the above ultrafiltration fraction with the best lipid-lowering effect in double-distilled water to prepare a solution with a concentration of 25 - 30 mg / mL, separate by Sephadex LH-20 column chromatography (2.0 × 100 cm), elute with double-distilled water, the flow rate is 0.5 - 0.6 mL / min, according to the gel chromatography chromatogram at 214 nm, collect the components of each chromatographic peak, measure the effects of each chromatographic peak component on lipid accumulation in the HepG2 cell model, select the chromatographic peak component with the best effect of reducing lipid accumulation, lyophilize to obtain the lipid-lowering gel chromatography enzymatic hydrolysate of large yellow croaker scales; RP-HPLC procedure: The above-mentioned gel filtration enzymatic hydrolysate of large yellow croaker scales was prepared into a solution with a concentration of 90-100 μg / mL using double-distilled water, and purified by RP-HPLC. According to the lipid-lowering activity of the prepared polypeptide, one polypeptide with high lipid-lowering activity was obtained. After determination, its sequence was: Ser-His-Gly-Pro-Cys-Phe (SHGPCF); the RP-HPLC conditions were as follows: injection volume 150-200 μL; chromatographic column Hypersil 300A C18 (250 mm×10.0 mm, 10 μm); mobile phase: 60% acetonitrile; elution rate 1.5-2.0 mL / min; ultraviolet detection wavelength 214 nm.

3. The application of the hexapeptide from Pseudosciaena crocea scales according to claim 1 in the preparation of lipid-lowering drugs.

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