Preparation method of collagen and application of collagen in anti-aging

Abstract

The invention provides a preparation method of collagen and an application of the collagen in anti-aging. According to the invention, the anti-aging antioxidant collagen peptide F-5-B is obtained through enzymolysis and column separation identification. It is identified that the polypeptide has good DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and lipid peroxidation reaction inhibiting capacity, the collagen peptide F-5-B can further inhibit melanoma cells from synthesizing melanin, and the polypeptide can further obviously increase the SOD level in the liver of a mouse. SOD can help the body resist attack of free radicals, so that the aging process is slowed down. Therefore, the anti-aging pharmaceutical composition prepared from the polypeptide has a relatively good application prospect.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a method for preparing collagen and its application in anti-aging. Background Art

[0002] Human aging is the result of multiple factors, including genetics, environment, and lifestyle. The main mechanisms include: Oxidative stress and free radical damage: free radicals produced by cell metabolism exceed antioxidant capacity, leading to damage to DNA, proteins, and cell membranes, accelerating the aging process. Cell aging and DNA damage: cell division accumulates DNA damage, repair capacity decreases, telomere shortening limits cell proliferation, and ultimately leads to tissue degeneration. Endocrine and metabolic disorders: decreased secretion of hormones such as gonads and thyroid glands, insulin resistance, and accumulation of metabolic waste (such as lipofuscin) destroy cell function. Immune system decline: thymus atrophy leads to decreased T cell production, increased chronic inflammation and autoimmune reactions, and weakened body defenses. Environmental and lifestyle factors: ultraviolet radiation, smoking, high-sugar diets, etc. accelerate oxidative stress and inflammatory reactions, shortening healthy life span.

[0003] Current research shows that the core mechanism of aging at the cellular level is that aging cells (such as fibroblasts and immune cells) decline in function, secrete pro-inflammatory factors (such as IL-6), form a "senescence-associated secretory phenotype" (SASP), and accelerate the aging of surrounding tissues. From a molecular level, it is protein glycosylation (AGEs), mitochondrial dysfunction, and imbalanced gene expression (such as decreased activity of the longevity gene SIRTUIN family) that lead to decreased metabolic and repair capabilities. From the organ system level, it is mainly microcirculatory disorders, decreased organ reserve function (such as heart, kidney) and neurodegeneration (such as brain atrophy) that cause multi-system coordinated aging.

[0004] Of course, aging is not untreatable. Common treatments include lifestyle intervention diet: low-sugar, low-fat, high-antioxidant diet (such as blueberries, deep-sea fish), supplementation of NAD+ precursors (NMN), resveratrol, etc. Exercise: a combination of aerobic and strength training to improve mitochondrial function and muscle mass. Work and rest: regular sleep (7-8 hours / day) to reduce oxidative stress. Currently, drug treatment is also an effective way. Including the use of antioxidants: vitamin E, coenzyme Q10 to reduce oxidative damage; hormone replacement: supplementation of estrogen, testosterone, etc. under the guidance of a doctor to relieve menopausal symptoms; photoelectric therapy: Thermage, photorejuvenation to improve skin sagging and pigmentation. Of course, there are also cutting-edge therapies, including stem cell therapy: umbilical cord mesenchymal stem cells can repair skin, ovaries and other tissues, and delay organ aging. Immune cell infusion: remove senescent cells (such as macrophages) and reduce inflammatory responses. Gene editing: targeted repair of longevity genes (such as FOXO3) or inhibition of aging-related pathways (such as mTOR).

[0005] One of the most popular anti-aging drugs currently being studied is human collagen. At present, the application of human collagen in anti-aging has become a research hotspot in the current biomedical and cosmetology fields. As the main component of the extracellular matrix (ECM), collagen accounts for 70%-80% of the dry weight of the skin. The integrity of its structure and function is directly related to the elasticity, barrier function and aging process of the skin. With age, the rate of collagen synthesis decreases (1%-1.5% loss per year after the age of 25), resulting in collagen fiber breakage, skin sagging and wrinkle formation.

[0006] Collagen interacts with fibroblasts, macrophages, etc. through its triple helix structure, rebuilding the ECM microenvironment and promoting collagen regeneration and repair. Collagen is also one of the most abundant proteins in the human body, with biological activities such as support, repair, and moisturizing, and has significant potential in the field of anti-aging. The hydroxyl and hydrophilic groups in the collagen molecule can bind a large amount of water, and still retain 45% of its own weight in water at a relative humidity of 70%. The mesh structure it forms can repair the skin barrier, reduce water loss, and provide a moist environment for cell metabolism.

[0007] For example, recombinant humanized type III collagen can improve skin barrier tolerance, improve redness and fine lines, and 28-day human tests show that skin elasticity and glossiness are significantly improved. Its antioxidant properties can also scavenge free radicals and delay photoaging. Recombinant collagen (such as Wei Yimei) is used to fill static wrinkles (nasolabial folds, tear grooves), improve skin quality, and enhance barrier function in joint photoelectric projects, with both support and regeneration effects. Animal-derived collagen (such as Shuangmei) requires skin testing due to potential allergic risks, while humanized products have better biocompatibility. Small molecule collagen peptides (2000-3000 Daltons) can penetrate into the dermis, promote collagen synthesis, and improve dryness, roughness and pigmentation. Skin care products containing recombinant type III collagen can repair epidermal damage and enhance skin elasticity. In addition, oral collagen peptides are absorbed through the intestines and stimulate endogenous collagen production, but high-purity, low-molecular-weight products must be selected to improve bioavailability. Compared with traditional anti-aging ingredients (such as retinol and phosphodiesterone), recombinant collagen has low immunogenicity, no irritation and excellent biological activity. Humanized type III collagen is 100% homologous to human collagen through precise replication of gene sequences. There is no rejection reaction after injection and the effect is cumulative. When combined with photoelectric therapy, collagen can synergistically enhance the skin's ability to repair and shorten the recovery period.

[0008] At present, the preparation method of human collagen mainly relies on gene recombination technology, combined with fermentation engineering and purification process. For example, through genetic engineering technology, human collagen genes (such as type I and type III) are cloned into expression vectors and transferred into host cells (such as Escherichia coli and yeast). For example, Swiss Weizhenhui uses genetic engineering technology, fermentation engineering technology and protein separation and purification technology to establish an efficient expression system for recombinant collagen. Chinese scientists have provided a theoretical basis for gene sequence design by analyzing the atomic structure of human type III collagen (PDB6A0A, 6A0C). Fermentation is the core link in the production of recombinant collagen. Depending on the use (medical devices or cosmetics), the process standards vary: Medical devices: GMP standards must be followed, high-density fermentation processes must be used, and temperature, pH and dissolved oxygen parameters must be controlled to ensure protein expression and stability. Cosmetics: Focusing on cost and efficiency, yeast secretion expression systems are commonly used, proteases are inactivated by heat treatment (60-65°C, 10-30 minutes), and salting out or chromatography purification is combined.

[0009] Although human collagen has made significant progress in the field of anti-aging, large-scale production still needs to further optimize the fermentation process and purification efficiency to reduce production costs. The preparation of collagen peptides with better effects is still the focus of current research. Summary of the invention

[0010] Oxidative stress is one of the main causes of cell aging, and the antioxidant components in yellow crucian carp are like "scavengers" in our body, helping us to remove the "garbage" in the body and maintain the health and vitality of cells. On this basis, the present invention extracts and prepares antioxidant collagen peptides from the skin of yellow crucian carp.

[0011] Specifically, the antioxidant collagen peptide is F-5-B, and its amino acid sequence is shown in SEQ ID NO:1.

[0012] The present invention also provides a type of polypeptide fragment obtained by deleting amino acids in the amino acid sequence of polypeptide SEQ ID NO: 1, which may be a polypeptide fragment in which amino acid deletion is performed at the amino terminus, carboxyl terminus or within the amino acid sequence of polypeptide SEQ ID NO: 1, respectively; or amino acid deletion is performed at both the amino terminus and carboxyl terminus of the amino acid sequence of polypeptide SEQ ID NO: 1; or amino acid deletion is performed at both the amino terminus and within the sequence of polypeptide SEQ ID NO: 1; or amino acid deletion is performed at both the carboxyl terminus and within the sequence of polypeptide SEQ ID NO: 1; or amino acid deletion is performed at both the amino terminus and within the sequence of polypeptide SEQ ID NO: 1; or amino acid deletion is performed at both the amino terminus, carboxyl terminus and within the sequence of polypeptide SEQ ID NO: 1.

[0013] In some embodiments, the number of amino acid deletions described in the present invention is within 7 amino acids, including 7 amino acids, such as 1, 2, 3, 4, 5, 6, 7 amino acids are deleted.

[0014] In the present invention, it can be the product of amino acid replacement or addition to the polypeptide sequence, or it can be the product of chemical modification of the amino, carboxyl, thiol, phenolic hydroxyl, imidazole, guanidine, indolyl, methylthio, etc. at the end of the main chain or side chain of the polypeptide molecule as the modification site.

[0015] The chemical modification described in the present invention is to chemically modify the polypeptide at the polypeptide level using appropriate modification methods and modifiers to improve the solubility, stability and half-life of the modified polypeptide drugs, which can be determined by those skilled in the art in a conventional manner.

[0016] More specifically, the present invention also provides a method for preparing collagen peptide F-5-B, wherein the protein peptide is obtained by enzymatically hydrolyzing collagen from fish skin and then screening and identifying a polypeptide with good antioxidant and anti-aging properties.

[0017] Furthermore, the present invention also provides an anti-aging pharmaceutical composition, wherein the composition contains an antioxidant collagen peptide F-5-B, whose amino acid sequence is shown in SEQ ID NO: 1.

[0018] Specifically, the pharmaceutical composition contains a pharmaceutically acceptable carrier.

[0019] Preferably, the dosage form of the pharmaceutical preparation includes tablets, capsules, oral liquids, granules or injections (such as freeze-dried powder injections). Pharmaceutically acceptable salts may be pharmacologically and pharmaceutically acceptable. Pharmacologically and pharmaceutically acceptable salts may be alkali metal salts or alkaline earth metal salts, preferably sodium salts, potassium salts, magnesium salts or calcium salts.

[0020] The pharmaceutical preparations involve enteral (e.g. oral, sublingual or rectal), parenteral or topical (e.g. transdermal pharmaceutical preparations) dosage forms. Organic or inorganic substances that do not react with the active ingredient can be used as carriers, such as water, oil, benzyl alcohol, polyethylene glycol, triacetin or other fatty acid glycerides, gelatin, lecithin, cyclodextrin, lactobiose or starch and other sugars, magnesium stearate, talc or cellulose. Oral medication is preferably tablets, dragees, capsules, powders, syrups, concentrates or drops, rectal administration is preferably suppositories, parenteral administration is preferably aqueous or oily solutions, or lyophilized agents. Suspensions, emulsions or implants can also be used, and topical medication can be used with patches or creams. Pharmaceutical preparations for parenteral use include sterile aqueous or anhydrous injections of active compounds, preferably solutions isotonic with the recipient's blood.

[0021] These pharmaceutical preparations may contain stabilizers, additives for controlling the release of pharmaceutically active compounds, antioxidants, buffers, antibacterial agents, and adjuvants for preparing isotonic solutions. Aqueous and anhydrous sterile suspensions may contain suspension additives and thickeners. Pharmaceutical preparations may be packaged in single-dose or multi-dose containers such as sealant bottles, or may be stored as freeze-dried products and, if necessary, may be formulated with sterile liquids such as water or saline solutions when used. Sterile powders, granules, or tablets may also be used in the same manner.

[0022] The pharmaceutical preparation can be used to prevent and treat aging-related diseases in humans and animals.

[0023] Furthermore, the pharmaceutically acceptable carrier is selected from at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, antioxidants, adsorbents, filter aids, and release retardants.

[0024] Preferably, the pharmaceutically acceptable carrier is selected from at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, polyethylene glycol, ethyl cellulose, liposome, methacrylic acid copolymer, polyvinyl acetate, carboxymethyl ethyl cellulose, carboxymethyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyacrylic acid resin, polycarboxyvinyl, alginate, carrageenan, carboxylic acid lactone, gum, polyvinyl alcohol, pregelatinized starch, cross-linked starch, sodium carboxymethyl starch, dextrin, polyethylene oxide, chitosan, chitosan, ion exchange resin and collagen.

[0025] Furthermore, the present invention also provides a pharmaceutical composition containing the polypeptide, which is a composition of the polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient, wherein the pharmaceutical excipient comprises an organic carrier and a pharmaceutical preparation excipient, and the polypeptide and the pharmaceutical preparation excipient form a composition, wherein the weight of the polypeptide is 1% to 50% of the total weight of the solid dispersion, and the weight of the excipient is 0.1% to 80% of the weight of the solid dispersion.

[0026] Beneficial Effects

[0027] The present invention provides a method for preparing collagen and its application in anti-aging. More specifically, the present invention obtains an anti-aging antioxidant collagen peptide F-5-B from fresh fish skin of yellow crucian carp through enzymatic hydrolysis and column separation and identification. It is identified that the polypeptide has good DPPH free radical scavenging ability, ABTS free radical scavenging ability and ability to inhibit lipid peroxidation reaction, and the collagen peptide F-5-B can also inhibit the ability of melanoma cells to synthesize melanin. The polypeptide can also significantly increase the level of SOD in mouse liver, and SOD can help the body resist the attack of free radicals, thereby slowing down the aging process. Therefore, the anti-aging pharmaceutical composition prepared by the polypeptide has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Effect of collagen peptide on melanin synthesis in melanoma cells

[0029] Figure 2 Effects of collagen peptide on exhaustive swimming time in mice DETAILED DESCRIPTION

[0030] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0031] Example 1 Preparation method of yellow crucian carp antioxidant collagen peptide

[0032] Select fresh fish skin of farmed yellow crucian carp, wash it thoroughly with running water, cut it into small pieces (0.5×0.5 cm), use 0.1M NaOH at a ratio of 1:10 (w / v), change the solvent every 5 hours, and then wash it with cold distilled water until neutral to remove non-collagen and other foreign proteins; then soak it with 10% n-butanol at a ratio of 1:10 (w / v) for 12 hours, change the solvent every 3 hours, and then rinse it with 10 times the volume of cold distilled water for better fat removal; soak it in about 85% ethanol for 12 hours, change the solvent every 3 hours, and finally rinse it with cold distilled water. After the pretreated fish skin is homogenized, trypsin, pepsin and alkaline protease are used for enzymolysis in sequence, wherein the amount of enzyme added is 4.5% of the fish skin mass, and the product is hydrolyzed for 4 hours to obtain a hydrolysis product; the hydrolysis product is then placed in a 95°C water bath for 10 minutes to kill enzymes to obtain an enzymolysis solution; activated carbon and the enzymolysis solution are mixed at a solid-to-liquid ratio of 0.02 (i.e., 2 grams of activated carbon are added to every 100ml of the enzymolysis solution) to obtain a mixed solution; the mixed solution is stirred at 300 rev / mins for 20 minutes to remove the fishy smell and decolorize, and then a plate and frame filter is used to filter, and a filter cloth is 180 meshes to obtain a decolorized solution; the decolorized solution is ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 10kD, and the filtrate is collected and freeze-dried at-30°C. The lyophilized powder was further separated and purified by Sephadex G-25 chromatography column (2.6 cm × 60 cm). The sample concentration was 20 mg / mL, the sample volume was 2 mL, and it was eluted with deionized water at a rate of 1.5 mL / min. The UV spectrophotometer was used for detection at 280 nm. The F-5 elution peak with the best antioxidant properties was collected for concentration and freeze-drying. A 1 mg / mL sample solution was prepared with 0.1% TFA deionized water, filtered through a 0.22 μm microporous membrane, and filtered using RP -HPLC was further separated and purified, and the chromatographic analysis conditions were as follows: injection volume, 25 μL; column type, C18 Hypersil BDS (250 mm × 4.6 mm, 5 μm); column temperature, 30 ° C; mobile phase was acetonitrile-water solution (containing 0.1 mL / 100 mL of trifluoroacetic acid), gradient elution was used, and the acetonitrile concentration was linearly increased from 0% to 100% within 50 min elution time; elution flow rate, 1.0 mL / min; UV detection wavelength was set at 280 nm. Similarly, after collecting each elution peak, the component F-5-B with the strongest antioxidant activity was selected through activity screening, concentrated and freeze-dried, and then the amino acid sequence of the peptide was identified using tandem mass spectrometry (MS / MS) and amino acid sequence analyzer, and the amino acid sequence of the antioxidant collagen peptide was identified as shown in SEQ ID NO: 1. The antioxidant collagen peptide was artificially synthesized and used for later use.

[0033] Example 2 Identification of the antioxidant properties of the antioxidant collagen peptide F-5-B

[0034] (1) Determination of DPPH free radical scavenging ability

[0035] The antioxidant peptides were studied by DPPH (1,1-Diphenyl-2-picryl-hydrazyl) free radical scavenging rate assay. The concentration was 1×10 -5 mol / L DPPH ethanol solution, stored in the dark. Add 2mL, 0.1mM DPPH anhydrous ethanol solution to a clean test tube containing 2mL of antioxidant collagen peptide F-5-B of different concentrations and mix well. After standing at room temperature for 30 minutes, the absorbance was measured at 517nm. The smaller the absorbance value, the stronger the free radical scavenging ability. Scavenging rate (%) = [1-{Ai-Aj} / A0], where A0 is 2mL, 0.1mM DPPH anhydrous ethanol solution + 2mL of sample solvent, blank control; Ai is 2mL, 0.1mM DPPH anhydrous ethanol solution + 2mL of sample; Aj is 2mL of anhydrous ethanol + 2mL of sample. The results are shown in Table 1.

[0036] Table 1 Free radical scavenging ability of antioxidant collagen peptide F-5-BDPPH

[0037] Group Clearance rate (%) F-5-B (50 μg / ml) 71.42±0.13 F-5-B (100 μg / ml) 83.96±0.25 F-5-B (200 μg / ml) 95.63±0.34 F-5-B (300 μg / ml) 98.89±0.58 Blank control - Vc (100 μg / ml) 81.46±0.38

[0038] - No scavenging ability was detected.

[0039] (2) Determination of ABTS free radical scavenging activity

[0040] Dissolve ABTS in deionized water to make the ABTS concentration reach 7mmol / L, and add potassium persulfate to make the concentration of potassium persulfate 2.45mmol / L. Then place the solution in the dark at room temperature overnight. Dilute the generated ABTS free radical solution with phosphate buffer (PBS, 0.2mol / L, pH7.4) to make its absorbance at 734nm 0.70. Take 0.1ml of polypeptide and mix it with 2.9ml ABTS free radical solution, shake it for 30s, react in the dark for 10min, and then measure the absorbance of the reaction solution at 734nm. Use distilled water instead of hydrolyzate as blank. Clearance rate (%) = {A0-Aj} / A0, where A0 is the absorbance of the mixture of 2.9mL ABTS reagent and 0.1mL distilled water; Aj is the absorbance of 2.9mL ABTS reagent and 0.1mL polypeptide solution of different concentrations. The results are shown in Table 2.

[0041] Table 2 Free radical scavenging ability of antioxidant collagen peptide F-5-BABTS

[0042]

[0043]

[0044] - No scavenging ability was detected.

[0045] (3) Determination of anti-lipid peroxidation activity

[0046] Mix an equal volume of fresh egg yolk and phosphate buffer (pH 7.4, 0.1 mol / L), dilute 25 times before use and stir evenly with a magnetic stirrer. Add 2.4 ml of egg yolk dilution, 2.4 ml of 25 mmol / L FeSO 4 ·H 2 O, 200μL of polypeptide sample, mix well and place in a water bath at 37℃ for 4h, add 0.8ml50% trichloroacetic acid and 2mlTBA, mix well and place at 95℃ for 30min. After cooling to room temperature, centrifuge at 8000r / min for 20min, take the supernatant and measure the absorbance at 532nm. Distilled water is used instead of polypeptide solution as blank. Among them, inhibition rate (%) = {A0-Aj} / A0, where A0 is the absorbance of blank control group; Aj is the absorbance of sample group. The results are shown in Table 3.

[0047] Table 3 Inhibition rate of antioxidant collagen peptide F-5-B against liposomes

[0048] Group Inhibition rate (%) F-5-B (50 μg / ml) 53.15±0.16 F-5-B (100 μg / ml) 63.48±0.27 F-5-B (200 μg / ml) 72.59±0.31 F-5-B (300 μg / ml) 77.84±0.62 Blank control - Vc (100 μg / ml) 54.63±0.45

[0049] - No inhibitory capacity was detected.

[0050] From the results in Tables 1 to 3, it can be seen that the isolated F-5-B polypeptide has a strong antioxidant capacity. It has a strong ability to scavenge DPPH free radicals, ABTS free radicals and inhibit lipid peroxidation reactions, and has a good citation prospect.

[0051] Example 3 Effect of the collagen peptide F-5-B of the present invention on melanin synthesis in melanoma cells.

[0052] Melanoma cells B16-F10 (purchased from HZBIO, catalog number: HZC-50319) were cultured in RPMI1640 medium (containing 10% fetal bovine serum) at 5% CO 2 , and cultured in a carbon dioxide incubator at a constant temperature of 37°C. Melanoma cells in the exponential growth phase after subculturing were taken and the cell density was adjusted to 1×10 6 Each well of a six-well plate was inoculated with 100 μl of culture medium and incubated at 37°C with 5% CO 2After culturing in an incubator for 24 hours, the sample was added (the collagen peptide sample of the present invention was dissolved in DMEM culture medium and filtered with a 0.22μm filter) to make the final concentration reach 100μg / mL and 200μg / mL respectively. The positive control was 100μg / mL of Vc, and the control group was without other components. After culturing for 48 hours, the digested cells were resuspended in 1mL of culture medium and the cells were counted. After counting, the culture medium was centrifuged and discarded, and 1mL of 1mol / LNaOH solution containing 10% DMSO was added to lyse the cells, ultrasonically broken for 1min, and water bathed at 90℃ for 2h. The A value was measured at 450nm on an enzyme reader to calculate the relative content of cell melanin. In this experiment, a culture medium without the collagen peptide of the present invention was also set as a blank control, and 3 replicates were set for each group. The relative content of melanin synthesis is calculated as follows: relative content of melanin synthesis / % = [(A value of treatment group / cell density of treatment group) / (A value of control group / cell density of control group)] × 100%, and the results are shown in Figure 1 .

[0053] Depend on Figure 1 It can be seen that the collagen peptide of the present invention can significantly inhibit the melanin synthesis of melanoma cells. Compared with the control group, as the concentration of the polypeptide increases, the collagen peptide of the present invention can gradually reduce the relative content of melanin in melanoma cells. When the concentration is 200 μg / mL, the relative content of melanin synthesis is (56.32±2.03)%, which is better than the reduction effect of the positive control group.

[0054] Example 4 Evaluation of the anti-fatigue effect of collagen peptide F-5-B of the present invention

[0055] 40 SPF male mice, body weight (20±2.5) g, feeding temperature (25±2)℃, relative humidity of air 55%~75%, mice were free to eat. After one week of adaptive feeding, mice were randomly divided into 3 groups according to body weight, blank control group (CK), F-5-B peptide feeding group, positive control group, 10 mice in each group. Gavage was performed once a day for 5 consecutive weeks. The CK group was gavaged with sterile saline, and the positive control group was gavaged with 0.5 mg / 20 g of Ganoderma lucidum polysaccharide (source leaf, B24477); the F-5-B peptide feeding group was gavaged with 0.5 mg / 20 g of polypeptide, and the gavage volume was 0.2 mL.

[0056] On the last day of the experiment, 1 hour after the mice were gavaged, a weighted swimming test was performed on the mice. The remaining 5 mice in each group were selected and weighed separately. A lead block weighing 5% of the body weight was tied to the base of the mouse's tail. The mice were placed in a constant temperature swimming box with a water temperature of 30°C and a water depth of 50 cm. During the experiment, a glass rod was used to gently stir the mice so that the mice's limbs were always in a swimming state. The time from the start of the mouse's swimming to the time when the head sank below the water surface for 7 seconds and could not float to the surface was recorded. This was the mouse's exhaustive swimming time. Exhaustive swimming time is the most important indicator that reflects the mouse's ability to resist exercise fatigue. The longer the exhaustive swimming time, the stronger the mouse's ability to resist exercise fatigue. The results are as follows Figure 2 shown.

[0057] from Figure 2 It can be seen that compared with the CK group, the F-5-B peptide can significantly increase the exhaustive swimming time after feeding, which reaches (956.4±37.5)s, which is more significant than the positive control group. This fully shows that the F-5-B peptide can effectively enhance the vitality of the body and achieve the anti-aging effect.

[0058] The liver tissues of mice in each group were homogenized, and the corresponding MDA and SOD activities were detected using kits. The SOD activity was detected according to the instructions of the total superoxide dismutase (T-SOD) assay kit; the MDA activity was detected according to the instructions of the malondialdehyde (MDA) assay kit. The results are shown in Table 4.

[0059] Table 4 Antioxidant properties of mice in each group

[0060]

[0061] # indicates P<0.05, the difference is significant; ## indicates P<0.01, the difference is extremely significant.

[0062] From the results in Table 4, it can be seen that the polypeptide of the present invention can significantly increase the level of SOD in the liver of mice, and SOD can help the body resist the attack of free radicals, thereby slowing down the aging process. In addition, the polypeptide of the present invention can significantly reduce the level of MDA in the liver of aging mice, and the anti-aging effect is significant.

[0063] Although the embodiments of the present invention are described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

Claims

1. An antioxidant collagen peptide is F-5-B, characterized in that The amino acid sequence of the peptide is shown in SEQ ID NO:

1.

2. An antioxidant and anti-aging pharmaceutical composition, characterized in that The antioxidant collagen peptide according to claim 1 is F-5-B.

3. Use of the antioxidant collagen peptide F-5-B according to claim 1 in the preparation of an antioxidant and anti-aging pharmaceutical composition.

4. The use according to claim 3, wherein the pharmaceutical composition contains a pharmaceutically acceptable carrier.

5. The use according to claim 4, wherein the dosage form of the pharmaceutical composition comprises tablets, capsules, oral liquids, granules or injections.

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