CN116082443A - Tuna fish scale oligopeptide and preparation method and application thereof - Google Patents
Tuna fish scale oligopeptide and preparation method and application thereof Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
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- A61P17/16—Emollients or protectives, e.g. against radiation
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- A61P17/18—Antioxidants, e.g. antiradicals
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to the technical field of biology, in particular to a tuna fish scale oligopeptide with skin ultraviolet injury repairing effect, and a preparation method and application thereof; according to the invention, tuna scales are used as raw materials, and oligopeptide Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) with skin ultraviolet injury repairing effect is obtained through pretreatment, composite enzymolysis, membrane ultrafiltration and chromatographic separation and purification, and the molecular weight of ESI-MS is 862.0Da. The oligopeptide YSGPLGIR with the skin ultraviolet injury repairing effect prepared by the invention can obviously improve the survival rate and the antioxidant enzyme activity of ultraviolet injury mouse fibroblasts (NIH 3T 3), and can be applied to preparing medicaments and health care products related to skin ultraviolet injury.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a tuna fish scale oligopeptide with skin ultraviolet injury repairing effect, and a preparation method and application thereof.
Background
The skin, which is the largest organ of the human body, is exposed to various harmful environments from the outside. Ultraviolet (UV) radiation in the environment that is too intense or too long can induce significant increases in the levels of reactive oxygen species (Reactive oxygen species, ROS) in skin tissue, which in turn can cause oxidative stress, leading to skin roughening, sagging, dryness, desquamation, wrinkling, pigmentation, etc., and severe can induce skin cancer. Thus, skin damage caused by UV is also known as skin Photoaging (Photoaging). Based on the above, attention is increasingly paid to the use of the anti-oxidative functional molecules, which are screened from the skin photoaging mechanism, in related products to eliminate or reduce the damage of ultraviolet rays to the skin.
Tuna is one of important operation fish species in the world ocean fishery, and scraps accounting for about 50-70% of the total weight are produced in the processing process, and mainly comprise tuna skin, viscera, minced meat, fish heads, fish scales and the like, so that waste of tuna resources is caused, and great pressure is brought to the ecological environment. Therefore, partial researches focus on comprehensive and efficient utilization of tuna offal to prepare functional molecules with medical health care value. Based on the above, the applicant uses the tuna processing byproduct-fish phosphorus as a raw material, and uses an enzymolysis process and a chromatographic preparation technology to prepare the oligopeptide with the skin ultraviolet injury repairing effect, and the oligopeptide can be used for preparing medicines or health-care products for treating skin photoaging.
Disclosure of Invention
The invention provides a tuna fish scale oligopeptide with skin ultraviolet injury repair effect, which can be used for preparing a medicine or a health-care product for treating skin photoaging. .
The oligopeptide is an octapeptide compound, the amino acid sequence of the octapeptide compound is Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR), and the molecular weight of the octapeptide compound is 862.0Da by ESI-MS.
A preparation method of the tuna scale oligopeptide comprises the following steps:
1) Pretreatment of tuna scales: adding tuna scales into NaOH solution, soaking for 6-8 hours at 20-25 ℃ to remove non-collagen; repeatedly washing the processed tuna scales with distilled water for 3-5 times, draining, adding EDTA-Na solution with pH of 7.4, soaking for 6-8 h (1 EDTA solution is replaced every 2 h) at room temperature, washing with distilled water for 3 times, drying and crushing;
2) Enzymolysis of tuna scales: adding the tuna scale powder into phosphate buffer solution with pH of 2.0, regulating the temperature of the solution to 35-45 ℃, regulating the pH value to 1.0-2.0, adding pepsin, hydrolyzing for 2-4 h, and inactivating enzyme at 95 ℃ for 15 min; regulating the temperature of the solution to 45-50 ℃, regulating the pH value to 6.5-8.0, adding neutral protease, reacting for 2-4 hours, inactivating enzyme at 95 ℃ for 15min, cooling to normal temperature, centrifuging at 9000rmp for 25-30 min, and collecting supernatant, namely tuna fish scale enzymatic hydrolysate;
3) Preparing oligopeptides of tuna scales: classifying the tuna scale enzymatic hydrolysate by an ultrafiltration membrane with the molecular weight cut-off of 1kDa, 5kDa and 10kDa, collecting the classified components, measuring the influence of the classified components on the cell viability of an ultraviolet-damaged mouse fibroblast (NIH 3T 3) model, selecting the component with the highest cell viability for freeze-drying to obtain a tuna scale ultrafiltration hydrolysate, and purifying the ultrafiltration hydrolysate by macroporous resin, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the tuna scale oligopeptide with the skin ultraviolet damage repairing effect;
in some embodiments of the invention, the tuna of step 1) is bonito Katsuwonus pelamis.
In some embodiments of the present invention, the concentration of the NaOH solution in the step 1) is 0.1mol/L, and the weight-volume ratio of the tuna scales to the NaOH solution is 1 g:10-15 mL.
In some embodiments of the invention, the concentration of EDTA-Na solution in the step 1) is 0.5mol/L, and the weight-volume ratio of the tuna scales to the EDTA-Na solution is 1 g:8-10 mL.
In some embodiments of the invention, the weight-to-volume ratio of the tuna scales to the phosphate buffer in the step 2) is 1 g:8-10 mL.
In some embodiments of the invention, the pepsin added in step 2) is 1.5-2.0% of the weight of the tuna scales.
In some embodiments of the invention, the amount of the neutral white enzyme added in the step 2) is 1.5-2.0% of the weight of the tuna scales.
In some embodiments of the invention, the macroporous resin, gel column chromatography and RP-HPLC purification of step 3) is performed by:
macroporous resin: dissolving the tuna scale ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 40-45 mg/mL, slowly adding the solution into a pretreated D101 macroporous resin column for chromatography, eluting with double distilled water, 30% ethanol and 95% ethanol with the volume of 3-5 times of column volume respectively, collecting 3 elution components with the flow rate of 1.0-1.5 mL/min, measuring the influence effect of the 3 components on the NIH3T3 survival rate of ultraviolet injury, selecting the component with the highest cell survival rate, and freeze-drying to obtain the tuna scale macroporous resin zymolyte.
Gel column chromatography: dissolving the tuna scale macroporous resin zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, separating by Sephadex G-15 column chromatography, eluting by using phosphate buffer solution at the flow rate of 0.6-0.9 mL/min, preparing a gel chromatography chromatogram according to the absorbance at 225nm, collecting each chromatographic peak, measuring the influence effect of each chromatographic peak component on the NIH3T3 survival rate of ultraviolet injury, selecting the chromatographic peak component with the highest cell survival rate, and freeze-drying to obtain the tuna scale gel chromatography zymolyte.
RP-HPLC purification: preparing 45-50 mug/mL of solution of the tuna scale gel chromatography zymolyte by double distilled water, purifying by RP-HPLC, and obtaining 1 high-activity polypeptide Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) according to the influence of the prepared oligopeptide on the survival rate of NIH3T3 damaged by ultraviolet rays, wherein the molecular weight of ESI-MS is 862.0Da.
Further, the RP-HPLC conditions are: the sample injection amount is 12-15 mu L; column Diamond C 18 (250 mm. Times.4.6 mm,5 μm); mobile phase: 55% acetonitrile; the elution speed is 0.9-1.2 mL/min; the ultraviolet detection wavelength is 225nm.
On the other hand, the invention provides application of the tuna fish scale oligopeptide YSGPLGIR in preparing a medicine or a health product for treating skin photoaging.
Compared with the prior art, the tuna fish scale oligopeptide YSGPLGIR with the skin ultraviolet injury repair effect provided by the invention can obviously improve the survival rate of ultraviolet injury mouse fibroblasts (NIH 3T 3) and the activity of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)) at the concentration of 10 mu M, reduce the content of lipid peroxide Malondialdehyde (MDA), and show obvious protection effect on ultraviolet injury cells; by utilizing mouse photodamage skin model research, YSGPLGIR can obviously raise the contents of hydroxyproline (Hyp), SOD, catalase (CAT), GSH-Px and matrix metalloproteinase inhibitor-1 (TIMP-1) in skin tissue, obviously reduce MDA and H 2 O 2 And matrix metalloproteinase (MMP-9) content, exhibit significant skin photodamage protection. Therefore, tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) has the advantages of safety, no toxic or side effect, strong skin photodamage protection effect and the like, and can be used for preparing medicaments or health-care products for treating skin photoaging.
Drawings
FIG. 1 shows the effect of the enzymatic hydrolysate (TSH), ultrafiltration fractions (TSH 1-TSH 4) and macroporous resin separation fractions (TSH 1-I-TSH 1-III) of tuna scale in the examples of the present invention on the survival rate of UV-damaged mouse fibroblasts (NIH 3T 3) at a concentration of 5 mg/mL.
FIG. 2 is a Sephadex G-15 chromatographic chart of a Sephadex according to an embodiment of the present invention.
FIG. 3 is a graph showing the effect of the preparation of the dextran gel Sephadex G-15 of the present invention on the survival rate of ultraviolet damaged mouse fibroblasts (NIH 3T 3) at a concentration of 5 mg/mL. .
FIG. 4 is a diagram of RP-HPLC analysis of an enzymatic hydrolysate prepared from Sephadex G-15 as an example of the present invention.
FIG. 5 shows the effect of RP-HPLC fractions (TSP 1-TSP 7) on the viability of UV-damaged mouse fibroblasts (NIH 3T 3) at a concentration of 5mg/mL according to an embodiment of the invention.
FIG. 6 is a mass spectrum of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) according to the invention.
FIG. 7 is a block diagram of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) according to the present invention.
FIG. 8 shows the effect of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) on the activity of antioxidant enzymes (SOD and GSH-Px) in ultraviolet damaged mouse fibroblasts (NIH 3T 3) according to the invention.
FIG. 9 is the effect of Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) of the present invention on Malondialdehyde (MDA) content in UV-injured mouse fibroblasts (NIH 3T 3).
FIG. 10 shows the effect of the levels of hydroxyproline (Hyp), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and matrix metalloproteinase inhibitor-1 (TIMP-1) in skin tissue of light-injured mice according to the invention.
FIG. 11 shows hydrogen peroxide (H) in light-damaged mouse skin tissue according to example Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) 2 O 2 ) And the effect of matrix metalloproteinase (MMP-9) content.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings. The solvent used in the present invention is not particularly limited, and commercially available conventional solvents can be used.
In cell experiments:
blank groups refer to: mouse fibroblasts (NIH 3T 3) cultured normally.
The model set refers to: mouse fibroblasts (NIH 3T 3) were irradiated with ultraviolet light (UVA, wavelength 320-400 nm) for 1 h.
Positive control group: mouse fibroblasts (NIH 3T 3) were first irradiated with ultraviolet (UVA, wavelength 320-400 nm) for 1h, and then treated with 10. Mu.M vitamin C for 24h.
YSGPLGIR group: mouse fibroblasts (NIH 3T 3) were first irradiated with ultraviolet (UVA, wavelength 320-400 nm) for 1h, then treated with 10. Mu.M oligopeptide YSGPLGIR for 24h.
In animal experiments:
blank groups refer to: normal ICR mouse skin.
The model set refers to: ICR mouse skin damaged by ultraviolet (uva+uvb) radiation.
Positive control group: after vitamin E treatment, the ultraviolet (UVA+UVB) radiation damaged ICR mouse skin.
YSGPLGIR group: the oligopeptide YSGPLGIR treatment was followed by ultraviolet (uva+uvb) radiation of damaged ICR mouse skin.
A tuna fish scale oligopeptide with skin ultraviolet injury repairing effect is prepared by the following steps: tuna fish scales, pretreatment, enzymolysis, ultrafiltration, macroporous resin column chromatography, gel filtration chromatography, high performance liquid chromatography preparation, fish scale oligopeptide with skin ultraviolet injury repair effect, and function evaluation.
The method comprises the following specific steps:
1) Pretreatment of tuna scales: adding the tuna scales into NaOH solution (0.10 mol/L) according to the feed liquid ratio of 1g to 12mL, soaking for 7h at 23 ℃ and removing non-collagen; repeatedly washing the processed tuna scales with distilled water for 4 times, draining, adding EDTA-Na solution (0.5 mol/L) with pH of 7.4 according to the ratio of feed liquid to 1 g/9 mL, soaking at room temperature for 8h (1 EDTA solution is replaced every 2 h), washing with distilled water for 3 times, drying, and pulverizing;
2) Enzymolysis of tuna scales: adding the tuna fish scale powder into phosphate buffer solution with pH of 2.0 according to the feed liquid ratio of 1g to 9mL, regulating the pH value of the solution to 1.5, regulating the temperature to 37 ℃, adding pepsin accounting for 1.6% of the weight of the tuna fish scale powder, hydrolyzing for 3h, and inactivating enzyme at 95 ℃ for 15 min; regulating the temperature of the solution to 48 ℃, regulating the pH value to 7.2, adding neutral protease accounting for 1.8% of the weight of the powder of the tuna scales, reacting for 4 hours, inactivating enzyme at 95 ℃ for 15 minutes, cooling to normal temperature, centrifuging at 9000rmp for 28 minutes, and collecting supernatant, namely tuna scales enzymatic hydrolysate;
3) Preparing oligopeptides of tuna scales: and (3) grading the tuna scale enzymatic hydrolysate by an ultrafiltration membrane with the molecular weight cut-off of 1kDa, 5kDa and 10kDa, collecting grading components TSH1 (MW <1 kDa), TSH2 (1 kDa < MW <5 kDa), TSH3 (5 kDa < MW <10 kDa) and TSH4 (MW >10 kDa), and determining the influence effect of the 4 components on the survival rate of ultraviolet-damaged mouse fibroblasts (NIH 3T 3) (see figure 1), wherein the TSH1 component has the strongest capability of promoting the cell survival rate, and freeze-drying the tuna scale ultrafiltration enzymatic hydrolysate. TSH1 is purified by macroporous resin column chromatography, gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) in sequence to obtain tuna fish scale oligopeptide with skin ultraviolet injury repairing effect, the molecular weight of the oligopeptide is determined by utilizing mass spectrum, and the amino acid sequence of the oligopeptide is determined by utilizing an amino acid sequence analyzer, wherein the specific process is as follows:
(1) d101 macroporous resin column chromatography: dissolving TSH1 in double distilled water to prepare a solution with the concentration of 40mg/mL, slowly adding a pretreated D101 macroporous resin column (5.0X100 cm) for chromatography, eluting with double distilled water, 30% ethanol and 95% ethanol with the volume of 4 times of the column volume under the condition of the flow rate of 1.5mL/min, collecting eluent to obtain 3 eluting components TSH1-I, TSH1-II and TSH1-III, measuring the influence effect of the 3 components on the survival rate of NIH3T3 damaged by ultraviolet rays (see figure 1), wherein the capability of the TSH1-III component for promoting the survival rate of cells is strongest, and freeze-drying to obtain the tuna fish scale macroporous resin zymolyte.
(2) Gel chromatography: dissolving the TSH1-III in double distilled water to prepare a solution with the concentration of 28mg/mL, separating by Sephadex G-15 column chromatography, eluting by double distilled water, collecting elution components TG-1-TG-3 (see figure 2) according to an absorbance curve at 225nm, determining the influence effect of the elution components on the survival rate of NIH3T3 damaged by ultraviolet rays (see figure 3), wherein the TG-2 component has the strongest capability of promoting the cell survival rate, and determining that TG-2 is a gel chromatography zymolyte.
(3) Refining by high performance liquid chromatography: the TG-2 fraction was prepared into a 50. Mu.g/mL solution with double distilled water, and purified by RP-HPLC (sample injection amount: 15. Mu.L; column chromatography Diamond C) 18 (250 mm. Times.4.6 mm,5 μm); mobile phase: 55% acetonitrile; elution rate 0.9 mL/min), 1 highly active oligopeptide (TSP 4) was obtained based on absorbance curve at 225nm (see fig. 4) and effect on the survival rate of NIH3T3 damaged by ultraviolet light (see fig. 5).
(4) And (3) structural detection: the chromatographic peak TSP4 which has the strongest promotion on the survival rate of NIH3T3 damaged by ultraviolet rays is collected, the sequencing requirement is met through RP-HPLC detection, the molecular weight is 862.0Da (see FIG. 6) by using ESI-MS, and the amino acid sequence is Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) by using a protein/polypeptide sequence analyzer (see FIG. 7).
(5) Functional evaluation: see [ Yuan Huijie, liao Ziqiong, ouyang Daofu, li Xiaomin, sun Changlei. Protection of aloe vera gel against fibroblast uv radiation damage [ J ]. University of Zhongshan university report (Nature science edition), 2018, 57 (2): 155-159] establishes a model of ultraviolet-damaged mouse fibroblasts (NIH 3T 3), and studies the protective effect of YSGPLGIR on ultraviolet-damaged cells, and results prove that: the tuna oligopeptide YSGPLGIR can obviously improve the survival rate of the NIH3T3 damaged by ultraviolet rays (see figure 5) and the activity of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)) and reduce the content of lipid peroxide Malondialdehyde (MDA) (see figure 9), and shows obvious protective effect on the NIH3T3 damaged by ultraviolet rays.
See [ Lu Qiujing, zhou Meng, wang Zhele, liu Liping. Protection effect of Dendrobium officinale extract on mice skin photodamage [ J ]]. Chinese patent medicine 2016, 38 (1): 2303-2306]Establishing a mouse photodamage skin model, and researching the protection effect of YSGPLGIR on the mouse photodamage by adopting a smearing administration mode, wherein the result proves that: YSGPLGIR can be significantly improvedThe contents of hydroxyproline (Hyp), SOD, catalase (CAT), GSH-Px and matrix metalloproteinase inhibitor-1 (TIMP-1) in skin tissue (see figure 10) significantly reduce MDA and H 2 O 2 And matrix metalloproteinase (MMP-9) content (see FIG. 11), exhibit significant skin photodamage protection. Therefore, tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) has the advantages of safety, no toxic or side effect, strong skin photodamage protection effect and the like, and can be used for preparing medicaments or health-care products for treating skin photoaging.
Finally, it should be noted that the above list is only one embodiment of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (10)
1. The tuna fish scale oligopeptide is characterized in that the oligopeptide is an octapeptide compound, the amino acid sequence of the octapeptide compound is Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR), and the molecular weight of the octapeptide compound measured by ESI-MS is 862.0Da.
2. The method for preparing the tuna scale oligopeptide according to claim 1, which is characterized by comprising the following steps:
1) Pretreatment of tuna scales: adding tuna scales into NaOH solution, soaking for 6-8 hours at 20-25 ℃ to remove non-collagen; repeatedly washing the processed tuna scales with distilled water for 3-5 times, draining, adding EDTA-Na solution with pH of 7.4, soaking for 6-8 h (1 EDTA solution is replaced every 2 h) at room temperature, washing with distilled water for 3 times, drying and crushing;
2) Enzymolysis of tuna scales: adding the tuna scale powder into phosphate buffer solution with pH of 2.0, regulating the temperature of the solution to 35-45 ℃, regulating the pH value to 1.0-2.0, adding pepsin, hydrolyzing for 2-4 h, and inactivating enzyme at 95 ℃ for 15 min; regulating the temperature of the solution to 45-50 ℃, regulating the pH value to 6.5-8.0, adding neutral protease, reacting for 2-4 hours, inactivating enzyme at 95 ℃ for 15min, cooling to normal temperature, centrifuging at 9000rmp for 25-30 min, and collecting supernatant, namely tuna fish scale enzymatic hydrolysate;
3) Preparing oligopeptides of tuna scales: classifying the tuna scale enzymatic hydrolysate by an ultrafiltration membrane with the molecular weight cut-off of 1kDa, 5kDa and 10kDa, collecting the classified components, measuring the influence of the classified components on the cell survival rate of an ultraviolet-damaged mouse fibroblast (NIH 3T 3) model, selecting the component with the highest cell survival rate for freeze-drying to obtain the tuna scale ultrafiltration enzymatic hydrolysate, and purifying the ultrafiltration enzymatic hydrolysate by macroporous resin, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the tuna scale oligopeptide with the skin ultraviolet damage repairing effect.
3. The method for producing a fish scale oligopeptide of tuna according to claim 2, wherein the tuna in step 1) is bonito Katsuwonus pelamis; the concentration of the NaOH solution in the step 1) is 0.1mol/L, and the weight-volume ratio of the tuna scales to the NaOH solution in the step 1) is 1 g:10-15 mL; the concentration of the EDTA-Na solution in the step 1) is 0.5mol/L, and the weight-volume ratio of the tuna scales to the EDTA-Na solution in the step 1) is 1 g:8-10 mL.
4. The method for preparing the oligopeptides according to claim 2, wherein the weight-to-volume ratio of the tuna scales to the phosphate buffer solution in the step 2) is 1 g:8-10 mL.
5. The method for preparing the oligopeptides according to claim 2, wherein the amount of pepsin added in the step 2) is 1.5-2.0% of the weight of the tuna scales.
6. The method for preparing oligopeptides according to claim 2, wherein the amount of the neutral protease added in the step 2) is 1.5-2.0% of the weight of the tuna scales.
7. The method for preparing the tuna scale oligopeptide according to claim 2, wherein the specific process of the macroporous resin in the step 3) is as follows: dissolving the tuna scale ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 40-45 mg/mL, slowly adding the solution into a pretreated D101 macroporous resin column for chromatography, eluting with double distilled water, 30% ethanol and 95% ethanol with the volume of 3-5 times of the column volume respectively, collecting 3 eluting components at the flow rate of 1.0-1.5 mL/min, measuring the effect of the 3 components on the survival rate of NIH3T3 damaged by ultraviolet rays, selecting the component with the highest cell survival rate, and freeze-drying to obtain the tuna scale macroporous resin zymolyte.
8. The method for preparing the tuna scale oligopeptide according to claim 2, wherein the specific gel column chromatography process of the step 3) is as follows: dissolving the tuna scale macroporous resin zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, separating by Sephadex G-15 column chromatography, eluting by using phosphate buffer solution at the flow rate of 0.6-0.9 mL/min, preparing a gel chromatography chromatogram according to the absorbance at 225nm, collecting each chromatographic peak, measuring the influence effect of each chromatographic peak component on the NIH3T3 survival rate of ultraviolet injury, selecting the chromatographic peak component with the highest cell survival rate, and freeze-drying to obtain the tuna scale gel chromatography zymolyte.
9. The method for preparing the tuna scale oligopeptide according to claim 2, wherein the specific process of large RP-HPLC purification in step 3) is as follows: preparing 45-50 mug/mL solution of tuna scale gel chromatography zymolyte by double distilled water, purifying by RP-HPLC, obtaining 1 high activity polypeptide Tyr-Ser-Gly-Pro-Leu-Gly-Ile-Arg (YSGPLGIR) according to the influence of the prepared oligopeptide on the survival rate of NIH3T3 damaged by ultraviolet rays, wherein the molecular weight of ESI-MS is 862.0Da, and the conditions of RP-HPLC are that: the sample injection amount is 12-15 mu L; column Diamond C 18 (250 mm. Times.4.6 mm,5 μm); mobile phase: 55% acetonitrile; the elution speed is 0.9-1.2 mL/min; the ultraviolet detection wavelength is 225nm.
10. Use of the tuna scale oligopeptide YSGPLGIR according to claim 1 for the preparation of a medicament or health food for the treatment of skin photoaging.
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