A tuna fish scale oligopeptide, its preparation method and application

Abstract

The present invention relates to the field of biotechnology, specifically to a tuna scale oligopeptide with the efficacy of repairing skin ultraviolet damage, its preparation method and application. The present invention uses tuna scales as raw materials, and through pretreatment, composite enzymatic hydrolysis, membrane ultrafiltration, and chromatographic separation and purification, an oligopeptide Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) with the efficacy of repairing skin ultraviolet damage is obtained. The molecular weight measured by ESI-MS is 862.0 Da. The oligopeptide YSGPLGIR prepared by the present invention with the efficacy of repairing skin ultraviolet damage can significantly improve the survival rate and antioxidant enzyme activity of ultraviolet-damaged mouse fibroblasts (NIH 3T3), and can be applied to the preparation of drugs and health products related to the treatment of skin ultraviolet damage.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a tuna scale oligopeptide with the efficacy of repairing skin ultraviolet damage, a preparation method thereof, and an application thereof. Background Art

[0002] As the largest organ of the human body, the skin is exposed to various harmful external environments. Excessive or prolonged ultraviolet (UV) radiation in the environment can induce a significant increase in the level of reactive oxygen species (ROS) in skin tissues, thereby causing oxidative stress, resulting in rough, loose, dry, peeling, wrinkled, pigmented skin, etc. In severe cases, skin cancer may be induced. Therefore, skin damage caused by UV is also called photoaging. Based on this, screening suitable antioxidant functional molecules from the mechanism of skin photoaging and applying them to related products to eliminate or reduce the damage of ultraviolet rays to the skin has increasingly attracted the attention of scholars.

[0003] Tuna is one of the important fishing species in the world's ocean fisheries. During the processing process, about 50% - 70% of the by-products are generated, mainly including tuna fish skin, internal organs, minced meat, fish heads, and fish scales, etc., which causes waste of tuna resources and brings great pressure to the ecological environment. Therefore, some studies focus on comprehensively and efficiently using tuna by-products to prepare functional molecules with medical and health care value. Based on this, the applicant uses tuna processing by-products - fish scales as raw materials, and uses enzymatic hydrolysis technology and chromatographic preparation technology to prepare an oligopeptide with the efficacy of repairing skin ultraviolet damage, and this oligopeptide can be used to prepare drugs or health care products for treating skin photoaging. Summary of the Invention

[0004] The present invention provides a tuna scale oligopeptide with the efficacy of repairing skin ultraviolet damage, and this oligopeptide can be used to prepare drugs or health care products for treating skin photoaging.

[0005] A tuna scale oligopeptide, which is an octapeptide compound, and its amino acid sequence is Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR), and the molecular weight measured by ESI-MS is 862.0 Da.

[0006] A preparation method of a tuna scale oligopeptide, comprising the following steps:

[0007] 1) Pretreatment of tuna scales: Add tuna scales to NaOH solution and soak at 20 - 25°C for 6 - 8 h to remove non - collagen proteins. After treatment, wash the tuna scales with distilled water repeatedly for 3 - 5 times, drain, add them to EDTA - Na solution with pH 7.4, soak at room temperature for 6 - 8 h (change the EDTA solution every 2 h), wash with distilled water 3 times, dry and pulverize;

[0008] 2) Enzymatic hydrolysis of tuna scales: Add the above - mentioned tuna scale powder to phosphate buffer solution with pH 2.0, adjust the temperature of the solution to 35 - 45°C, adjust the pH value to 1.0 - 2.0, add pepsin, hydrolyze for 2 - 4 h, inactivate the enzyme at 95°C for 15 min; adjust the temperature of the solution to 45 - 50°C, adjust the pH value to 6.5 - 8.0, add neutral protease, react for 2 - 4 h, inactivate the enzyme at 95°C for 15 min, cool to room temperature, centrifuge at 9000 rmp for 25 - 30 min, and collect the supernatant, namely the enzymatic hydrolysate of tuna scales;

[0009] 3) Preparation of tuna scale oligopeptides: Subject the enzymatic hydrolysate of tuna scales to ultrafiltration through ultrafiltration membranes with molecular weight cut - offs of 1 kDa, 5 kDa, and 10 kDa for fractionation, collect the fractionated components, determine their effects on the cell survival rate of the ultraviolet - damaged mouse fibroblast (NIH3T3) model, select the fraction with the highest cell survival rate and freeze - dry it to obtain the ultrafiltered enzymatic hydrolysate of tuna scales. This ultrafiltered enzymatic hydrolysate is successively purified by macroporous resin, gel column chromatography, and reverse - phase high - performance liquid chromatography (RP - HPLC) to obtain tuna scale oligopeptides with the efficacy of repairing skin ultraviolet damage;

[0010] In some embodiments of the present invention, the tuna in step 1) is Katsuwonus pelamis.

[0011] In some embodiments of the present invention, the concentration of the NaOH solution in step 1) is 0.1 mol / L, and the weight - to - volume ratio of the tuna scales to the NaOH solution is 1 g:10 - 15 mL.

[0012] 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 tuna scales to the EDTA - Na solution is 1 g:8 - 10 mL.

[0013] In some embodiments of the present invention, the weight - to - volume ratio of the tuna scales to the phosphate buffer solution in step 2) is 1 g:8 - 10 mL.

[0014] In some embodiments of the present invention, the addition amount of pepsin in step 2) is 1.5 - 2.0% of the weight of the tuna scales.

[0015] In some embodiments of the present invention, the addition amount of neutral protease in step 2) is 1.5-2.0% of the weight of tuna scales.

[0016] In some embodiments of the present invention, the specific processes of macroporous resin, gel column chromatography and RP-HPLC purification in step 3) are as follows:

[0017] Macroporous resin: Dissolve the above-mentioned ultrafiltration hydrolysate of tuna scales in double-distilled water to form a solution with a concentration of 40-45 mg / mL, slowly add it to the pretreated D101 macroporous resin column chromatography, and elute with 3-5 column volumes of double-distilled water, 30% ethanol, and 95% ethanol respectively, with a flow rate of 1.0-1.5 mL / min. Collect 3 elution fractions, measure the effects of the 3 fractions on the survival rate of NIH 3T3 cells damaged by ultraviolet rays, select the fraction with the highest cell survival rate, and lyophilize to obtain the macroporous resin hydrolysate of tuna scales.

[0018] Gel column chromatography: Dissolve the above-mentioned macroporous resin hydrolysate of tuna scales in double-distilled water to form a solution with a concentration of 25-30 mg / mL, separate it by Sephadex G-15 column chromatography of dextran gel, and elute with phosphate buffer solution, with a flow rate of 0.6-0.9 mL / min. Make a gel chromatography chromatogram according to the absorbance at 225 nm, collect each chromatographic peak, measure the effects of each chromatographic peak fraction on the survival rate of NIH 3T3 cells damaged by ultraviolet rays, select the chromatographic peak fraction with the highest cell survival rate, and lyophilize to obtain the gel column chromatography hydrolysate of tuna scales.

[0019] RP-HPLC purification: Dissolve the above-mentioned gel column chromatography hydrolysate of tuna scales in double-distilled water to form a solution with a concentration of 45-50 μg / mL, and purify it by RP-HPLC. According to the effect of the prepared oligopeptide on the survival rate of NIH 3T3 cells damaged by ultraviolet rays, obtain 1 highly active polypeptide Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR), and the molecular weight is determined to be 862.0 Da by ESI-MS.

[0020] Furthermore, the RP-HPLC conditions are as follows: injection volume 12-15 μL; chromatographic column Diamonsil C 18 (250 mm × 4.6 mm, 5 μm); mobile phase: 55% acetonitrile; elution speed 0.9-1.2 mL / min; ultraviolet detection wavelength 225 nm.

[0021] On the other hand, the present invention provides the application of the above-mentioned tuna scale oligopeptide YSGPLGIR in the preparation of drugs or health products for treating skin photoaging.

[0022] Compared with the prior art, the tuna scale oligopeptide YSGPLGIR with the efficacy of repairing skin ultraviolet damage provided by the present invention can significantly improve the survival rate of ultraviolet-damaged mouse fibroblasts (NIH 3T3) and the activities of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)) at a concentration of 10 μM, and reduce the content of lipid peroxide malondialdehyde (MDA), showing a significant protective effect on ultraviolet-damaged cells; the study using a mouse photo-damaged skin model proves that YSGPLGIR can significantly increase the contents of hydroxyproline (Hyp), SOD, catalase (CAT), GSH-Px and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the skin tissue, and significantly reduce the contents of MDA, H2O2 and matrix metalloproteinase (MMP-9), showing a significant protective effect on skin photo-damage. Therefore, Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) has the advantages of being safe, non-toxic and having strong protective effect on skin photo-damage, and can be used to prepare drugs or health care products for treating skin photo-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows the effects of the tuna scale enzymolysis solution (TSH), ultrafiltration components (TSH1-TSH4) and macroporous resin separation components (TSH1-I-TSH1-III) of the examples of the present invention on the survival rate of ultraviolet-damaged mouse fibroblasts (NIH3T3) at a concentration of 5 mg / mL.

[0024] Figure 2 is the chromatogram of Sephadex G-15 gel filtration chromatography of the examples of the present invention.

[0025] Figure 3 shows the effects of the Sephadex G-15 prepared enzymolysate components of the examples of the present invention on the survival rate of ultraviolet-damaged mouse fibroblasts (NIH 3T3) at a concentration of 5 mg / mL..

[0026] Figure 4 is the RP-HPLC analysis chart of the Sephadex G-15 prepared enzymolysate of the examples of the present invention.

[0027] Figure 5 shows the effects of the RP-HPLC separation components (TSP1-TSP7) of the examples of the present invention on the survival rate of ultraviolet-damaged mouse fibroblasts (NIH 3T3) at a concentration of 5 mg / mL.

[0028] Figure 6It is the mass spectrometry diagram of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) in the embodiment of the present invention.

[0029] Figure 7 It is the structural diagram of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) in the embodiment of the present invention.

[0030] Figure 8 It is the influence of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) in the embodiment of the present invention on the activities of antioxidant enzymes (SOD and GSH-Px) in ultraviolet-damaged mouse fibroblasts (NIH 3T3).

[0031] Figure 9 It is the influence of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) in the embodiment of the present invention on the content of malondialdehyde (MDA) in ultraviolet-damaged mouse fibroblasts (NIH 3T3).

[0032] Figure 10 It is the influence of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) in the embodiment of the present invention on the contents of hydroxyproline (Hyp), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in photo-damaged mouse skin tissue.

[0033] Figure 11 It is the influence of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) in the embodiment of the present invention on the contents of hydrogen peroxide (H2O2) and matrix metalloproteinase (MMP-9) in photo-damaged mouse skin tissue. Specific embodiments

[0034] The present invention will be further described in detail below in conjunction with the embodiments with reference to the drawings. There is no particular limitation on the solvents used in the present invention, and commercially available conventional solvents can be adopted.

[0035] In cell experiments:

[0036] The blank group refers to: mouse fibroblasts (NIH 3T3) cultured normally.

[0037] The model group refers to: mouse fibroblasts (NIH3T3) irradiated with UVA (ultraviolet A, wavelength 320 - 400 nm) for 1 h.

[0038] Positive control group: Mouse fibroblasts (NIH 3T3) were first irradiated with ultraviolet light (UVA, wavelength 320 - 400 nm) for 1 h, and then treated with 10 μM vitamin C for 24 h.

[0039] YSGPLGIR group: Mouse fibroblasts (NIH 3T3) were first irradiated with ultraviolet light (UVA, wavelength 320 - 400 nm) for 1 h, and then treated with 10 μM oligopeptide YSGPLGIR for 24 h.

[0040] In animal experiments:

[0041] The blank group refers to: The skin of normal ICR mice.

[0042] The model group refers to: The skin of ICR mice damaged by ultraviolet light (UVA + UVB) radiation.

[0043] Positive control group: The skin of ICR mice damaged by ultraviolet light (UVA + UVB) radiation after treatment with vitamin E.

[0044] YSGPLGIR group: The skin of ICR mice damaged by ultraviolet light (UVA + UVB) radiation after treatment with oligopeptide YSGPLGIR.

[0045] A tuna scale oligopeptide with the efficacy of repairing skin ultraviolet damage, the preparation process flow is as follows: Tuna scales → Pretreatment → Enzymolysis → Enzymolysate → Ultrafiltration → Macroporous resin column chromatography → Gel filtration chromatography → High performance liquid chromatography preparation → Scale oligopeptide with the efficacy of repairing skin ultraviolet damage → Functional evaluation.

[0046] The specific steps are as follows:

[0047] 1) Pretreatment of tuna scales: Add tuna scales to NaOH solution (0.10 mol / L) at a material-liquid ratio of 1 g:12 mL and soak at 23 °C for 7 h to remove non-collagen proteins; after treatment, the tuna scales are washed repeatedly with distilled water 4 times, drained, and added to EDTA-Na solution (0.5 mol / L) with a pH of 7.4 at a material-liquid ratio of 1 g:9 mL, and soaked at room temperature for 8 h (replace the EDTA solution every 2 h), then washed 3 times with distilled water, dried and pulverized;

[0048] 2) Enzymatic hydrolysis of tuna fish scales: Add the above-mentioned tuna fish scale powder to phosphate buffer solution with a pH of 2.0 according to the material-liquid ratio of 1 g:9 mL. Adjust the pH value of the solution to 1.5, adjust the temperature to 37 °C, add pepsin accounting for 1.6% of the weight of the tuna fish scale powder, hydrolyze for 3 h, and inactivate the enzyme at 95 °C for 15 min; adjust the temperature of the solution to 48 °C, adjust the pH value to 7.2, add neutral protease accounting for 1.8% of the weight of the tuna fish scale powder, react for 4 h, inactivate the enzyme at 95 °C for 15 min, cool to room temperature, centrifuge at 9000 rmp for 28 min, and collect the supernatant, which is the enzymatic hydrolysate of tuna fish scales;

[0049] 3) Preparation of tuna fish scale oligopeptides: The enzymatic hydrolysate of tuna fish scales is fractionated by ultrafiltration membranes with a molecular weight cut-off of 1 kDa, 5 kDa, and 10 kDa, and the fractionated components TSH1 (MW < 1 kDa), TSH2 (1 kDa < MW < 5 kDa), TSH3 (5 kDa < MW < 10 kDa), and TSH4 (MW > 10 kDa) are collected. The effects of the 4 components on the survival rate of ultraviolet-damaged mouse fibroblasts (NIH 3T3) are measured (see Figure 1 ). The TSH1 component has the strongest ability to promote cell survival rate. After freeze-drying, it is the ultrafiltration enzymatic hydrolysate of tuna fish scales. TSH1 is purified successively by macroporous resin column chromatography, gel column chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain tuna fish scale oligopeptides with the efficacy of repairing skin ultraviolet damage. Its molecular weight is measured by mass spectrometry, and its amino acid sequence is determined by an amino acid sequence analyzer. The specific process is as follows:

[0050] ① D101 macroporous resin column chromatography: Dissolve the above-mentioned TSH1 in double-distilled water to prepare a solution with a concentration of 40 mg / mL, and slowly add it to the pretreated D101 macroporous resin column (5.0 × 160 cm) for chromatography. Under the condition of a flow rate of 1.5 mL / min, elute with 4 column volumes of double-distilled water, 30% ethanol, and 95% ethanol respectively, and collect the eluate to obtain 3 elution components TSH1-I, TSH1-II, and TSH1-III. Measure the effects of the 3 components on the survival rate of ultraviolet-damaged NIH 3T3 (see Figure 1 ). The TSH1-III component has the strongest ability to promote cell survival rate. After freeze-drying, it is the macroporous resin enzymatic hydrolysate of tuna fish scales.

[0051] ② Gel chromatography: Dissolve the above-mentioned TSH1-III in double-distilled water to prepare a solution with a concentration of 28 mg / mL, and separate it by Sephadex G-15 column chromatography of dextran gel. Elute with double-distilled water, and collect the elution components TG-1 to TG-3 according to the absorbance curve at 225 nm (see Figure 2 ). Measure its effects on the survival rate of ultraviolet-damaged NIH 3T3 (see Figure 3) Among them, the TG-2 component has the strongest ability to promote cell survival rate, and TG-2 is determined as the gel filtration enzymatic hydrolysate.

[0052] ③ High-performance liquid chromatography purification: The above TG-2 component was prepared into a solution with a concentration of 50 μg / mL with double-distilled water and purified by RP-HPLC (sample injection volume 15 μL; chromatographic column Diamonsil C 18 (250 mm × 4.6 mm, 5 μm); mobile phase: 55% acetonitrile; elution speed 0.9 mL / min), and according to the absorbance curve at 225 nm (see Figure 4 ) and the effect on the survival rate of NIH 3T3 cells damaged by ultraviolet rays (see Figure 5 ), a highly active oligopeptide (TSP4) was obtained.

[0053] ④ Structure detection: The chromatographic peak TSP4 with the strongest promotion effect on the survival rate of NIH 3T3 cells damaged by ultraviolet rays was collected. After RP-HPLC detection, it met the sequencing requirements. The molecular weight was determined to be 862.0 Da by ESI-MS (see Figure 6 ), and the amino acid sequence was determined to be Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) by a protein / polypeptide sequence analyzer (see Figure 7 ).

[0054] ⑤ Function evaluation: Referring to the literature [Yuan Huijie, Liao Ziqiong, Ouyang Daofu, Li Xiaomin, Sun Changlei. Protective effect of aloe vera gel on ultraviolet radiation damage of fibroblasts [J]. Acta Scientiarum Naturalium Universitatis Sunyatseni, 2018, 57(2): 155-159], a model of ultraviolet-damaged mouse fibroblasts (NIH 3T3) was established to study the protective effect of YSGPLGIR on ultraviolet-damaged cells. The results showed that: tuna oligopeptide YSGPLGIR could significantly improve the survival rate of NIH3T3 cells damaged by ultraviolet rays (see Figure 5 ) and the activities of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)) (see Figure 8 ), and reduce the content of lipid peroxide malondialdehyde (MDA) (see Figure 9 ), showing a significant protective effect on NIH 3T3 cells damaged by ultraviolet rays.

[0055] Reference [Lu Qiujing, Zhou Mengdi, Wang Zhele, Liu Liping. Protective effect of Dendrobium officinale extract on photodamage of mouse skin [J]. Chinese Patent Medicine, 2016, 38(1): 2303-2306] A mouse skin model with photodamage was established, and the protective effect of YSGPLGIR on photodamage of mouse skin was studied by smear administration. The results showed that YSGPLGIR could significantly increase the contents of hydroxyproline (Hyp), SOD, catalase (CAT), GSH-Px and matrix metalloproteinase inhibitor-1 (TIMP-1) in skin tissue (see Figure 10 ), significantly reduced the content of MDA, H2O2 and matrix metalloproteinase (MMP-9) (see Figure 11 ), showing a significant protective effect against skin photodamage. Therefore, Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) has the advantages of being safe, non-toxic, and having a strong protective effect against skin photodamage, and can be used to prepare drugs or health products for treating skin photoaging.

[0056] 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 fish scale oligopeptide, characterized in that The oligopeptide is an octapeptide compound with the amino acid sequence Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR), and the molecular weight determined by ESI-MS is 862.0 Da.

2. Use of the tuna scale oligopeptide YSGPLGIR as described in claim 1 in the preparation of a medicament for treating skin photoaging.

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