An oligopeptide from tuna swim bladder for reducing blood lipid and antioxidation and its application

Abstract

The present invention discloses a tuna swim bladder hypolipidemic and antioxidant oligopeptide and its application. The present invention uses tuna swim bladder as a raw material, and after enzymatic hydrolysis, ultrafiltration, gel chromatography purification, and RP-HPLC purification, the tuna swim bladder hypolipidemic and antioxidant oligopeptide His-Ser-Gly-Pro-Tyr-Met (HSGPYM) is prepared, and its molecular weight is 690.8 Da. The HSGPYM of the present invention can significantly reduce the contents of triglyceride (TG) and total cholesterol (TC) in the lipid accumulation model of HepG2 cells, showing good lipid-lowering activity; at the same time, it can significantly increase the antioxidant enzyme level in the cell model, effectively scavenge excessive reactive oxygen species, and reduce oxidative stress damage. The HSGPYM of the present invention has the advantages of being safe, non-toxic, and having strong lipid-lowering and antioxidant capabilities, and can be used for the development of functional products for the treatment of non-alcoholic fatty liver disease, and can also be used as an additive for health products and foods.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a tuna swim bladder hypolipidemic and antioxidant oligopeptide and its application. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury with excessive fat accumulation (≥5% hepatocyte weight) and is closely related to insulin resistance (IR) and genetic susceptibility, including non-alcoholic simple fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFLD can not only lead to liver disease disability and death, but is also closely related to the high incidence of metabolic syndrome (MetS), type 2 diabetes (T2DM), cardiovascular diseases, colorectal tumors, etc. Moreover, chronic hepatitis B virus (HBV) infected patients often have NAFLD. It is estimated that about 25% of the global population suffered from NAFLD in 2018, among which 10%-20% was NASH, and the incidence of liver cirrhosis within 10 years of the latter was as high as 25%. With the rapid increase of obese and MetS populations in China, NAFLD has become the leading cause of the first chronic liver disease and abnormal liver biochemical indexes in health examinations in China, but there is no effective drug for the clinical treatment of NAFLD. The "two-hit" theory, that is, lipid deposition and oxidative stress injury, is an important theory to explain the pathogenesis of NAFLD. Therefore, finding therapeutic drugs targeting the molecular mechanisms (lipid deposition, oxidative damage) of the occurrence and development of NAFLD has become the focus of research. Summary of the Invention

[0003] The present invention uses tuna swim bladder as raw material, and prepares an oligopeptide with hypolipidemic and antioxidant functions by enzymatic hydrolysis process and chromatographic preparation technology. This oligopeptide is safe and has no toxic side effects, and can be used for the development of functional products for the treatment of non-alcoholic fatty liver disease, and can also be used as a safe additive for health products and foods.

[0004] The first technical problem to be solved by the present invention is to provide a tuna swim bladder hypolipidemic and antioxidant oligopeptide in view of the above technical status. The amino acid sequence of this oligopeptide is His-Ser-Gly-Pro-Tyr-Met (HSGPYM), and the molecular weight measured by ESI-MS is 690.8 Da.

[0005] The second technical problem to be solved by the present invention is to provide a preparation method of a tuna swim bladder hypolipidemic and antioxidant oligopeptide.

[0006] A preparation method of tuna swim bladder hypolipidemic and antioxidant oligopeptides, which comprises the following steps: using tuna swim bladder as raw material, and then preparing tuna swim bladder hypolipidemic and antioxidant oligopeptides through enzymolysis, ultrafiltration, gel chromatography purification, and RP-HPLC purification.

[0007] A preparation method of tuna swim bladder hypolipidemic and antioxidant oligopeptides specifically comprises the following steps:

[0008] 1) Pretreatment of tuna swim bladder: Thaw the tuna swim bladder, remove impurities, homogenize it into a paste in a tissue homogenizer, add it to NaOH solution (0.05mol / L), soak at 4°C for 18 - 24h, filter, rinse the NaOH with distilled water until clean, dry, add ethyl acetate, perform ultrasonic treatment at room temperature at 42KHZ and 300W for 25 - 30min, centrifuge to remove the supernatant, and obtain pretreated tuna swim bladder powder;

[0009] 2) Enzymolysis of tuna swim bladder: Take the above pretreated tuna swim bladder powder, add distilled water, adjust the temperature to 35 - 40°C, adjust the pH value to 1.0 - 2.0, add pepsin, hydrolyze for 4 - 5h, inactivate the enzyme at 95°C for 10min; when the sample cools to 50 - 55°C, adjust the pH value to 9.5 - 10.5, add alkaline protease, react for 4 - 5h, inactivate the enzyme at 95°C for 10min, cool to room temperature, centrifuge at 9000rmp for 18 - 20min, and collect the supernatant, namely tuna swim bladder enzymolysis solution;

[0010] 3) Preparation of tuna swim bladder hypolipidemic oligopeptides: Subject the tuna swim bladder enzymolysis solution to fractionation through ultrafiltration membranes with a molecular weight cut-off of 1kDa, 5kDa, and 10kDa, collect the fractionated components, detect the effects of each component on lipid accumulation and the content of reactive oxygen species (ROS) in the HepG2 cell model, and purify the ultrafiltration components with the best hypolipidemic and ROS scavenging effects through Sephadex LH-20 column chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) in sequence to obtain tuna swim bladder hypolipidemic oligopeptides.

[0011] In some embodiments of the present invention, the weight-to-volume ratio of tuna swim bladder to NaOH solution (0.05mol / L) in step 1) is 1g:18 - 20mL.

[0012] In some embodiments of the present invention, the weight-to-volume ratio of tuna swim bladder to ethyl acetate in step 1) is 1g:12 - 15mL.

[0013] In some embodiments of the present invention, the weight-to-volume ratio of pretreated tuna swim bladder powder to distilled water in step 2) is 1g:8 - 10mL.

[0014] In some embodiments of the present invention, the addition amount of pepsin in step 2) is 1.5-1.8% of the weight of the fish swim bladder powder.

[0015] In some embodiments of the present invention, the addition amount of alkaline protease in step 2) is 0.8-1.0% of the weight of the fish swim bladder powder.

[0016] In some embodiments of the present invention, the Sephadex LH-20 column chromatography step in step 3) is as follows: dissolve the above ultrafiltration component with the best lipid-lowering effect in double-distilled water to prepare a solution with a concentration of 35-45 mg / mL, separate it by Sephadex LH-20 column chromatography (2.6×120 cm), elute with double-distilled water, the flow rate is 0.6-0.9 mL / min, according to the gel chromatography chromatogram at 220 nm, collect the components of each chromatographic peak, measure the effects of each chromatographic peak component on lipid accumulation and ROS content in the HepG2 cell model, select the chromatographic peak component with the best effect of reducing lipid accumulation and scavenging ROS, freeze-dry to obtain the tuna fish swim bladder lipid-lowering gel chromatography enzymolysate.

[0017] In some embodiments of the present invention, the RP-HPLC purification step in step 3) is as follows: dissolve the above tuna fish swim bladder lipid-lowering gel chromatography enzymolysate in double-distilled water to prepare a solution with a concentration of 100-120 μg / mL, purify it by RP-HPLC, and obtain 1 high-activity oligopeptide His-Ser-Gly-Pro-Tyr-Met (HSGPYM) according to the lipid-lowering and ROS-scavenging abilities of the prepared polypeptide, and the molecular weight is determined by ESI-MS to be 690.8 Da.

[0018] In some embodiments of the present invention, the RP-HPLC purification conditions in step 3) are: the injection volume is 180-200 μL; the chromatographic column is Hypersil 300A C18 (250 mm×10.0 mm, 10 μm); the mobile phase is 55% acetonitrile; the elution speed is 1.5-2.0 mL / min; the ultraviolet detection wavelength is 220 nm.

[0019] Compared with the prior art, the tuna fish swim bladder lipid-lowering and antioxidant oligopeptide HSGPYM provided by the present invention can significantly reduce the contents of triglyceride (TG) and total cholesterol (TC) in the HepG2 cell lipid accumulation model at a concentration of 10 μM, showing good lipid-lowering activity; at the same time, it can significantly increase the antioxidant enzyme level in the cell model, effectively scavenge excessive reactive oxygen free radicals, and reduce oxidative stress damage. The His-Ser-Gly-Pro-Tyr-Met (HSGPYM) of the present invention has the advantages of being safe, non-toxic, and having strong lipid-lowering and antioxidant abilities, and can be developed into auxiliary treatment drugs and health foods for non-alcoholic fatty liver disease (NAFLD). Brief Description of the Drawings

[0020] Figure 1 It is a diagram showing the effect of the ultrafiltration fraction of the tuna swim bladder enzymolysis solution in the embodiment of the present invention on the lipid content in the HepG2 cell lipid accumulation model at a concentration of 5 mg / mL.

[0021] Figure 2 It is a diagram showing the effect of the ultrafiltration fraction of the tuna swim bladder enzymolysis solution in the embodiment of the present invention on the content of reactive oxygen species (ROS) in the HepG2 cell lipid accumulation model at a concentration of 5 mg / mL.

[0022] Figure 3 It is a chromatogram of Sephadex LH-20 chromatography in the embodiment of the present invention.

[0023] Figure 4 It is a diagram showing the effect of the prepared enzymolysis product fraction of Sephadex LH-20 in the embodiment of the present invention on the lipid content in the HepG2 cell lipid accumulation model at a concentration of 5 mg / mL.

[0024] Figure 5 It is a diagram showing the effect of the prepared enzymolysis product fraction of Sephadex LH-20 in the embodiment of the present invention on the content of reactive oxygen species (ROS) in the HepG2 cell lipid accumulation model at a concentration of 5 mg / mL.

[0025] Figure 6 It is an RP-HPLC analysis chromatogram of the prepared enzymolysis product of Sephadex LH-20 in the embodiment of the present invention.

[0026] Figure 7 It is a mass spectrum diagram of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention.

[0027] Figure 8 It is a structural diagram of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention.

[0028] Figure 9 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the lipid content in the HepG2 cell lipid accumulation model.

[0029] Figure 10 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the content of triglyceride (TG) in the HepG2 cell lipid accumulation model.

[0030] Figure 11 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the total cholesterol (TC) content in the lipid accumulation model of HepG2 cells.

[0031] Figure 12 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the superoxide dismutase (SOD) activity in the lipid accumulation model of HepG2 cells.

[0032] Figure 13 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the glutathione peroxidase (GSH-PX) activity in the lipid accumulation model of HepG2 cells.

[0033] Figure 14 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the reactive oxygen species (ROS) content in the lipid accumulation model of HepG2 cells.

[0034] Figure 15 It is a diagram showing the effect of His-Ser-Gly-Pro-Tyr-Met (HSGPYM) in the embodiment of the present invention on the viability of HepG2 cells. Detailed implementation manners

[0035] The following further describes the present invention in detail with reference to the embodiments of the accompanying drawings. However, they do not constitute a limitation or restriction on the scope of the present invention.

[0036] There is no particular limitation on the solvent used in the present invention, and commercially available conventional solvents can be used.

[0037] Normal group: HepG2 cells that grow in the culture medium without any treatment.

[0038] Model group: HepG2 cells treated with a fatty acid (oleic acid: palmitic acid 2:1) solution for 24 h.

[0039] Positive control group: HepG2 cells treated with simvastatin and a fatty acid (oleic acid: palmitic acid 2:1) solution for 24 h.

[0040] Sample group: HepG2 cells treated with the experimental sample and a fatty acid (oleic acid: palmitic acid 2:1) solution for 24 h.

[0041] Embodiment

[0042] 1) Pretreatment of tuna swim bladder: Thaw the tuna swim bladder, remove impurities, homogenize it into a paste in a tissue homogenizer, then add it to a NaOH solution (0.05 mol / L) at a solid-liquid ratio of 1 g:18 mL and soak it at 4°C for 24 h. Filter, rinse the NaOH with distilled water, and dry it. Add ethyl acetate at a ratio of 1 g:15 mL, perform ultrasonic treatment at room temperature at 42 KHZ and 300 W for 30 min, centrifuge to remove the supernatant, and obtain tuna swim bladder powder;

[0043] 2) Enzymatic hydrolysis of tuna swim bladder: Take the above-mentioned pretreated tuna swim bladder powder, add distilled water at a solid-liquid ratio of 1 g:8 mL, adjust the temperature to 38°C, adjust the pH value to 1.5, add 1.8% pepsin based on the weight of the swim bladder powder, hydrolyze for 5 h, and inactivate the enzyme at 95°C for 10 min. When the sample cools to 50°C, adjust the pH value to 10.0, add 1.0% alkaline protease, react for 4.5 h, inactivate the enzyme at 95°C for 10 min, cool to room temperature, centrifuge at 9000 rmp for 20 min, and collect the supernatant, namely tuna swim bladder enzymatic hydrolysate (TSPH);

[0044] 3) Preparation of lipid-lowering oligopeptides from tuna swim bladder: The tuna swim bladder enzymatic hydrolysate TSPH is fractionated by ultrafiltration membranes with molecular weight cut-offs of 1 kDa, 5 kDa, and 10 kDa, and the fractionated components TSPH-1 (MW < 1 kDa), TSPH-2 (1 kDa < MW < 5 kDa), TSPH-3 (5 kDa < MW < 10 kDa), and TSPH-4 (MW > 10 kDa) are collected. The effects of each component on lipid accumulation (the results are shown in Figure 1 ) and the content of reactive oxygen species (ROS) (the results are shown in Figure 2 ) in the HepG2 cell model are detected. TSPH-1 has the best lipid-lowering and ROS-scavenging effects. TSPH-1 is purified successively by Sephadex LH-20 column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain lipid-lowering and antioxidant oligopeptides from tuna swim bladder, and its structure is determined using an amino acid sequence analyzer and mass spectrometry. The specific process is as follows:

[0045] Sephadex LH-20 column chromatography: Dissolve the above-mentioned TSPH-1 in double-distilled water to prepare a solution with a concentration of 40 mg / mL, separate it by Sephadex LH-20 column chromatography (2.6 × 120 cm), elute with double-distilled water at a flow rate of 0.8 mL / min. According to the gel filtration chromatogram at 220 nm (see Figure 3 ), collect the components of each chromatographic peak (TSPH-1A to TSPH-1C), and measure the lipid accumulation of each chromatographic peak component in the HepG2 cell model (see Figure 4) and the effects on the content of reactive oxygen species (ROS) (see Figure 5 ), it was determined that TSPH-1C had the best effect on reducing lipid accumulation clearance and reactive oxygen species (ROS), namely the gel filtration enzymatic hydrolysate;

[0046] RP-HPLC purification: The above TSPH-1C was prepared into a solution with a concentration of 110 μg / mL with double-distilled water and purified by RP-HPLC (sample injection volume 180 μL; chromatographic column Hypersil 300AC18 (250 mm × 10.0 mm, 10 μm); mobile phase: 55% acetonitrile; elution speed 1.8 mL / min; ultraviolet detection wavelength 220 nm). According to the absorbance curve at 220 nm, 11 oligopeptide components TSP1-TSP11 were prepared (see Figure 6 ), and the lipid-lowering and reactive oxygen species (ROS)-scavenging activities of the prepared oligopeptide components were measured to obtain 1 highly active lipid-lowering oligopeptide (TSP9);

[0047] Structure detection: Collect TSP9 with the highest lipid-lowering activity. After RP-HPLC detection, it met the sequencing requirements. The amino acid sequence was determined to be His-Ser-Gly-Pro-Tyr-Met (HSGPYM) using a protein / polypeptide sequence analyzer (see Figure 8 ), and the molecular weight was determined to be 690.8 Da by ESI-MS (see Figure 7 ).

[0048] Functional evaluation: Referring to the literature [Cheng Jing, Liu Ying, Liu Yaojie, Zhao Jiang, Ji Yanglin, Liu Dong, Wang Hao. Caffeic acid phenethyl ester reduces free fatty acid-induced lipid accumulation in HepG2 cells by activating AMPK [J]. Food Science, 2020, 41(11): 171-178], a HepG2 cell lipid accumulation model was established to evaluate the lipid-lowering effect of the tuna swim bladder lipid-lowering hexapeptide His-Ser-Gly-Pro-Tyr-Met (HSGPYM). The results showed that: at a concentration of 10 μM, HSGPYM could significantly reduce lipid accumulation in the cell model and significantly reduce the contents of triglyceride (TG) and total cholesterol (TC) (see Figures 9 - 11 ); in addition, His-Ser-Gly-Pro-Tyr-Met (HSGPYM) could significantly increase the levels of antioxidant enzymes (SODG and SH-Px) in the cell model (see Figures 12 - 13 ), effectively scavenge excessive reactive oxygen species (ROS) (see Figure 14 ), reduce oxidative stress damage to cells, and had no significant effect on the viability of HepG2 cells (see Figure 15). Therefore, His-Ser-Gly-Pro-Tyr-Met (HSGPYM) has the advantages of safety, no toxic side effects, strong lipid-lowering and antioxidant abilities, and can be developed as an auxiliary treatment drug and health food for non-alcoholic fatty liver disease (NAFLD).

[0049] 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 tuna swim bladder hypolipidemic and antioxidant oligopeptide, characterized in that The amino acid sequence of the oligopeptide is His-Ser-Gly-Pro-Tyr-Met (HSGPYM), and the molecular weight measured by ESI-MS is 690.8 Da.

2. A preparation method of tuna swim bladder hypolipidemic and antioxidant oligopeptides, characterized in that The preparation method includes the following steps: using tuna swim bladder as the raw material, and then preparing the lipid-lowering and antioxidant oligopeptide from tuna swim bladder through enzymatic hydrolysis, ultrafiltration, gel chromatography purification, and RP-HPLC purification; specifically including the following steps: 1) Pretreatment of tuna swim bladder: Thaw the tuna swim bladder, remove impurities, and homogenize it into a paste in a tissue homogenizer, add it to 0.05 mol / L NaOH solution and soak it at 4 °C for 18 - 24 h, filter, rinse the NaOH with distilled water until clean, dry, add ethyl acetate, and perform ultrasonic treatment at room temperature at 42 KHZ and 300 W for 25 - 30 min, centrifuge to remove the supernatant to obtain the pretreated tuna swim bladder powder; 2) Enzymatic hydrolysis of tuna swim bladder: Take the above pretreated tuna swim bladder powder, add distilled water, adjust the temperature to 35 - 40 °C, adjust the pH value to 1.0 - 2.0, add pepsin, and hydrolyze for 4 - 5 h, inactivate the enzyme at 95 °C for 10 min; when the sample cools to 50 - 55 °C, adjust the pH value to 9.5 - 10.5, add alkaline protease, react for 4 - 5 h, inactivate the enzyme at 95 °C for 10 min, cool to room temperature, centrifuge at 9000 rpm for 18 - 20 min, and collect the supernatant, which is the enzymatic hydrolysate of tuna swim bladder; 3) Preparation of tuna swim bladder hypolipidemic oligopeptides: The tuna swim bladder enzymatic hydrolysate was fractionated by ultrafiltration membranes with molecular weight cut-offs of 1 kDa, 5 kDa, and 10 kDa. The fractionated components were collected, and the effects on lipid accumulation and the content of reactive oxygen species (ROS) in the HepG2 cell model were detected. The ultrafiltration fractions with the best hypolipidemic and ROS-scavenging effects were successively purified by Sephadex LH-20 column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain tuna swim bladder hypolipidemic oligopeptides. Among them, in step 1), the weight-volume ratio of tuna swim bladder to 0.05 mol / L NaOH solution is 1 g:18 - 20 mL; in step 1), the weight-volume ratio of tuna swim bladder to ethyl acetate is 1 g:12 - 15 mL; in step 2), the weight-volume ratio of pretreated tuna swim bladder powder to distilled water is 1 g:8 - 10 mL; in step 2), the addition amount of pepsin is 1.5 - 1.8% of the weight of the swim bladder powder; the addition amount of alkaline protease is 0.8 - 1.0% of the weight of the swim bladder powder; the Sephadex LH-20 column chromatography step in step 3) is as follows: The above ultrafiltration fraction with the best hypolipidemic effect was dissolved in double-distilled water to form a solution with a concentration of 35 - 45 mg / mL, and separated by Sephadex LH-20 column chromatography. The specifications of the Sephadex LH-20 column chromatography are 2.6×120 cm, eluted with double-distilled water at a flow rate of 0.6 - 0.9 mL / min. According to the gel chromatography chromatogram at 220 nm, the components of each chromatographic peak were collected, and the effects on lipid accumulation and the ROS content in the HepG2 cell model were determined. The chromatographic peak component with the best lipid accumulation reduction and ROS scavenging effects was selected, freeze-dried to obtain tuna swim bladder hypolipidemic gel chromatography enzymatic hydrolysate; the RP-HPLC purification step in step 3) is as follows: The above tuna swim bladder hypolipidemic gel chromatography enzymatic hydrolysate was made into a solution with a concentration of 100 - 120 μg / mL with double-distilled water, and purified by RP-HPLC. According to the hypolipidemic and ROS-scavenging abilities of the prepared polypeptide, 1 highly active oligopeptide His-Ser-Gly-Pro-Tyr-Met (HSGPYM) was obtained, and the molecular weight was determined by ESI-MS to be 690.8 Da; the RP-HPLC purification conditions in step 3) are: injection volume 180 - 200 μL; chromatographic column Hypersil 300A C18: 250 mm×10.0 mm, 10 μm; mobile phase: 55% acetonitrile; elution speed 1.5 - 2.0 mL / min; ultraviolet detection wavelength 220 nm.

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