Collagen-containing composition and application thereof in anti-aging

Abstract

The invention provides a composition containing collagen and an application of the composition in anti-aging, and particularly provides an anti-aging polypeptide KS-C-6, and the anti-aging polypeptide KS-C-6 has a good anti-oxidation characteristic and an anti-apoptosis characteristic. The composition prepared from the anti-aging polypeptide KS-C-6 and collagen can effectively improve the water content of the skin and has the anti-aging effect of treating the skin.

Description

Technical Field

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

[0002] The skin is the largest organ in the human body, responsible for many physiological functions such as protection, sensation, temperature regulation, secretion, excretion and immunity. However, due to environmental or radiation reasons, the skin ages rapidly.

[0003] One of the main causes of skin aging is photoaging, also known as external aging, which is premature aging of the skin caused by environmental influences (mainly ultraviolet exposure). The ultraviolet spectrum is divided into three categories: ultraviolet C (UVC: 200-280nm), ultraviolet B (UVB: 280-320nm) and ultraviolet A (UVA: 320-400nm). Among them, UVC has been absorbed in the atmosphere and cannot reach the ground. UVB can irradiate the epidermis and modify the DNA in the cells to form cyclobutane pyrimidine dimers, causing damage to the skin epidermis, dry skin, pigmentation, and flushing. UVA can penetrate the epidermis and reach the dermis. After long-term irradiation, it can damage fibroblasts, cause a decrease in collagen and fibrin, collapse of skin structure, and form wrinkles. Reactive oxygen species (ROS) produced by ultraviolet radiation can cause destructive oxidative stress, activate the arachidonic acid pathway and mediate inflammatory responses. Photoaging accelerates skin aging, and the skin becomes dry, rough, wrinkled, capillary dilation, and melanin deposition.

[0004] In addition, oxidative stress is one of the most important causes of the aging process. UV radiation can induce the production of ROS, leading to oxidative damage and accumulation of oxidation products, which are hallmarks of oxidative stress. Damage caused by ROS is one of the important mechanisms leading to skin aging. The sources of ROS in cells include enzymes and non-enzymes. Enzymes that produce reactive oxygen include the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidoreductase (XOR), several peroxisomal oxidases, cytochrome P450 family, cyclooxygenases and lipoxygenases. ROS can be produced not only in mitochondria through respiration, but also through the endoplasmic reticulum, cytosol, cell membrane, and peroxisome. According to the free radical theory of aging, ROS, which are mainly produced by oxidative cell metabolism, play an important role in both chronological aging and photoaging. ROS causes damage to skin cells and tissues. In particular, it destroys antioxidant enzymes such as glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT).

[0005] Conquering skin diseases by delaying the aging process of the skin has become one of the hot topics in current life science research. At present, collagen has been widely used to delay skin aging. In traditional Chinese medicine classics such as the Compendium of Materia Medica, there are many records of collagen products such as tortoise shell glue, donkey-hide glue, deer antler glue, turtle shell glue, and fish glue. Collagen products from salmon, jellyfish, sea cucumbers, etc. can improve skin quality. The potential ability of collagen to delay skin aging has also been reported abroad. In general, collagen is widely used in delaying skin aging.

[0006] Specifically, studies have shown that collagen peptides extracted from cod skin have good hygroscopic and moisturizing properties at different relative humidity levels, can reduce damage caused by ultraviolet radiation, and have multiple functions such as anti-aging and repairing damaged skin, and have great potential for application in moisturizing skin care products. In addition, palmitoyl peptides repair skin aging damage by adsorbing and interacting with keratin in the stratum corneum and hair stratum corneum. The innovative cosmeceutical ointment with the peptide is safe and non-irritating. In vivo studies on oyster protein hydrolysate peptides with a molecular weight range of 302.17 to 2936.43Da have shown that topical application on the skin can significantly reduce water loss, epidermal hyperplasia, and degradation of collagen and elastin fibers caused by chronic ultraviolet B irradiation, while protecting the skin from light damage and having good moisturizing and anti-wrinkle effects. Chitosan / collagen peptide nanoparticles stabilized Pickering emulsions with biocompatibility and biodegradation gradients have been rated as effective green biodegradable topical carriers. The moisturizing properties of the emulsion can be affected by changing the ratio of oil phase to water phase, which is more targeted to people with different skin types. Studies have shown that when sea cucumber collagen peptides were compared with tilapia and pig skin collagen peptides, the commercial value of sea cucumber industrial byproducts was higher, but all three collagen peptides showed better moisturizing and hygroscopicity than glycerol. In general, the moisturizing effects of collagen peptides from different sources and molecular weight distributions are slightly different. Compared with the hygroscopicity and antioxidant activity of collagen peptides from pig skin, cow skin, cod skin, and cow bones, donkey skin collagen peptides perform better. In addition, some synthetic collagen peptides have also been proven to have specific skin care, moisturizing and anti-aging effects, such as an innovative neuropeptide, acetyl hexapeptide-8, which is used in cosmetic formulas to not only enhance the moisturizing properties of the skin, but also reduce wrinkles caused by facial muscle contraction. Not only that, palmitoyl pentapeptide-3, which is obtained by combining natural collagen peptides with palmitic acid, can significantly improve skin affinity and water retention while effectively promoting the regeneration of skin collagen, hyaluronic acid and elastic fibers, thereby reducing fine lines on the skin and greatly enhancing the firmness and glossiness of the skin.

[0007] Although there are currently a variety of treatments for skin aging, compositions with better and more lasting therapeutic effects still need to be further developed and prepared, and there is a strong market demand. Summary of the invention

[0008] The invention screens and separates the anti-aging polypeptide KS-C-6 with good antioxidant properties from knife fish skin, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0009] Furthermore, the composition prepared by the anti-aging polypeptide KS-C-6 of the present invention and collagen has a better anti-aging effect and can be used to prepare an anti-aging pharmaceutical composition.

[0010] Specifically, the present invention also provides a pharmaceutical composition for treating anti-aging, which comprises the anti-aging polypeptide KS-C-6 of the present invention and collagen.

[0011] Specifically, the collagen is commercially available, or prepared by enzymatic separation from fish skin.

[0012] More specifically, the preparation method of the collagen is to take fresh knife fish skin, remove excess fish meat, cut it into small pieces of square with a side length of 0.5 cm with scissors, put it into 0.1mol / L NaOH according to a ratio of 1:30 (g / mL), stir it gently for 6 hours, change the solution every 2 hours, wash it with distilled water until it is neutral, drain it, and remove non-collagen. Then put the fish skin into a 10% butanol solution according to a ratio of 1:30 (g / mL), stir it gently for 24 hours, change the solution every 8 hours, wash it with distilled water until it is close to neutral, drain it, and remove excess fat. Then the fish skin treated is subjected to the optimized substrate concentration of 1:15, the temperature is 50°C, the pH is 6.0, the enzyme amount of papain is 3500U / g, and the treatment time is 5 hours. The collagen solution extracted by acid method was centrifuged with a refrigerated centrifuge to obtain the supernatant, sodium chloride was added to stand for salting out, and the precipitate was centrifuged to obtain the precipitate. The precipitate was re-dissolved with 1mol sodium chloride solution of 0.05mol / L Tris-HCl (pH=7.4) for 12h, and the supernatant was centrifuged to obtain the supernatant, and sodium chloride was used for secondary salting out. The supernatant was discarded after centrifugation, and the precipitate was re-dissolved with 0.5M acetic acid for 12h and dialyzed. The collagen precipitate obtained by salting out was soaked in 10 times deionized water at 4°C for several minutes. When the precipitate becomes transparent, it was centrifuged and the supernatant was sucked out, and it was redissolved in 0.5M acetic acid solution, slowly stirred to dissolve it, and placed in a dialysis bag for dialysis at 4°C. The molecular weight cutoff of the dialysis bag is 14000. The re-solution was dialyzed against 0.5M acetic acid, 0.1M acetic acid and deionized water respectively until it was dialyzed with 0.1M AgNO3. It was checked that there was no Cl in the external solution. -1 The dialysis was terminated and the collagen was obtained by freeze drying.

[0013] Specifically, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0014] Also provided are pharmaceutical compositions comprising the polypeptides of the invention. Such compositions comprise a therapeutically or prophylactically effective amount of a polypeptide or protein mixed with a pharmaceutically acceptable material and a physiologically acceptable formulation material. The pharmaceutical composition may comprise a formulation material for modifying, maintaining or retaining, for example, the pH, weight molar osmotic pressure concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides (preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients.

[0015] The property of the main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other materials common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or its suitable substitute. In one embodiment of the invention, the composition can be prepared for storage in the form of lyophilized mass or aqueous solution by mixing the selected composition with the desired purity with an optional formulation agent. In addition, the therapeutic composition can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0016] Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient and processing the resulting granular mixture (optionally after grinding) to obtain tablets or lozenge cores. Suitable adjuvants may be added as needed. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol and sorbitol; starch from corn, wheat, rice, potato or other plants; cellulose, such as methylcellulose, hydroxypropyl methylcellulose, or sodium carboxymethylcellulose; gums, including gum arabic and tragacanth; and proteins such as gelatin and collagen. Disintegrants or solubilizers may be added as needed, such as cross-linked polyvinyl pyrrolidone, agar and alginic acid or its salts, such as sodium alginate.

[0017] The route of administration of the pharmaceutical composition is according to known methods, such as oral, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional approach, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous or intraperitoneal injection; and intranasal, enteral, topical, sublingual, urethral, ​​vaginal or rectal methods, by sustained release system or by implant device. When necessary, the composition can be administered by push injection or continuous infusion or by implant device. Alternatively or additionally, the composition can be topically administered via implantation of a membrane, sponge or other suitable material that has been adsorbed or encapsulated with the desired molecule. When an implant device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be via diffusion, timed release pills or continuous administration.

[0018] The effective amount of the pharmaceutical composition used in treatment will depend, for example, on the treatment context and purpose. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will therefore vary in part depending on the delivered molecule, the indication for which the polypeptide is used, the route of administration, and the patient's size (weight, body surface or organ size) and condition (age and general health). Therefore, the clinician can gradually titrate the dose and modify the route of administration to obtain the best therapeutic effect. The general dose can be about 0.1 mg / kg-up to about 100 mg / kg or more, depending on the factors mentioned above. The polypeptide composition can preferably be injected or administered intravenously. Long-acting pharmaceutical compositions can be administered every 3-4 days, weekly or every 2 weeks, depending on the half-life and clearance rate of the specific formulation. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Generally, the composition is administered until a dose that achieves the desired effect is reached. The composition can therefore be administered as a single dose or multiple doses (at the same or different concentrations / dose) over time, or as a continuous infusion. Further improvements in appropriate doses are routinely performed. The appropriate dose can be determined by using appropriate dose-response data.

[0019] Beneficial Effects

[0020] The present invention provides a composition containing collagen and its application in anti-aging, and more specifically provides an anti-aging polypeptide KS-C-6, which has good antioxidant and anti-apoptotic properties. The anti-aging polypeptide KS-C-6 and collagen are prepared into a composition together, which can effectively improve the moisture content of the skin and treat the anti-aging effect of the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Anti-aging peptide KS-C-6 antioxidant properties identification results

[0022] Figure 2 Effects of each group on the content of hydroxyproline in skin tissue DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1 Identification of the Antioxidant Properties of the Anti-Aging Peptide KS-C-6

[0025] The amino acid sequence of the polypeptide KS-C-6 obtained by the applicant from the knife fish skin in the early stage is shown in SEQ ID NO: 1, and it was commissioned to be synthesized by Shanghai Qiangyao Biology. HFF-1 human skin fibroblasts (Zhongqiao Xinzhou, item number ZQ0450) were cultured in 1640 medium containing 10% fetal bovine serum and 1% double antibody at 37°C and 5% CO2. When the cell growth confluence reached more than 80%, the cells were passaged every 2 days.

[0026] HFF-1 cells in the logarithmic growth phase were selected and digested with 0.25% trypsin to adjust the cell concentration to 5 × 10 4 / mL, 100μL cell suspension per well was inoculated into a 96-well culture plate, and cultured in an incubator at 37℃ and 5% CO2 for 24h. Under an inverted microscope, the cells were completely attached to the wall and grew well. Carefully aspirate the old culture medium with a 1mL syringe. The sample group was added with a culture medium with an optimized concentration of 120μmol / L H2O2. After culturing for 24h, the culture medium was aspirated and culture medium containing low, medium and high concentrations of polypeptides was added (the concentrations of polypeptide KS-C-6 were 50μg / mL, 100μg / mL and 200μg / mL, respectively, and the positive control was 200μg / mL Vc). Blank culture medium was added to the blank control group. After culturing in an incubator at 37℃ and 5% CO2 for 24h, the cell proliferation rate was determined by CCK-8 method. Cell proliferation rate = (A1-A2) / (A3-A2)×100%, where A1 represents the absorbance of wells containing cells, CCK-8 solution and different concentrations of drugs; A2 represents the absorbance of wells containing CCK-8 solution but no cells or drugs; A3 represents the absorbance of wells containing cells, CCK-8 solution but no samples.

[0027] The protective ability of peptides against oxidative damage of fibroblasts is shown in Figure 1 ,Depend on Figure 1 It can be seen that as the concentration of the polypeptide increases, the polypeptide can effectively resist the damage of H2O2 to cells in a dose-dependent manner. Under the concentration condition of 200 μg / mL, the cell proliferation rate produced by the polypeptide of the present invention is (119.13±2.03)%, which is higher than the proliferation effect of the positive control, indicating that the polypeptide of the present invention has a better promoting effect.

[0028] Example 2 Detection of cell apoptosis rate by polypeptide

[0029] HFF-1 cells in the logarithmic growth phase were selected and digested with 0.25% trypsin to adjust the cell suspension concentration to 8 × 10 4 / mL, inoculated in 6-well culture plates, 1mL per well, placed in a 37°C, 5% CO2 incubator for 24 hours, observed under an inverted microscope, the cells were completely attached to the wall and in good growth condition. Carefully aspirate the old culture medium with a 1mL syringe, add culture medium containing (the concentrations of polypeptide KS-C-6 are 50μg / mL, 100μg / mL, 200μg / mL, and the positive control is 100μg / mL Vc), culture for 24 hours, use the culture medium without sample as blank control, use AnnexinVPE / 7-AAD apoptosis kit to pre-treat the cells, and detect by flow cytometry. The experiment was conducted in parallel 3 times. The results are shown in Table 1.

[0030] Table 1 Effects of each group on cell apoptosis rate

[0031] Group Apoptosis rate (%) Blank control group 3.55±0.13 50μg / mL peptide group 1.84±0.15 100μg / mL peptide group 1.63±0.10 200μg / mL peptide group 1.48±0.04 Positive control group 1.75±0.08

[0032] As shown in Table 1, compared with the blank control, both the peptide and the positive control can significantly reduce the apoptosis rate of skin fibroblasts. After the peptide was exposed to a concentration of 200 μg / mL, the apoptosis rate of the cells decreased from (3.55±0.13)% of the blank control to (1.48±0.04)%.

[0033] Example 3 Preparation of collagen

[0034] Take fresh knife fish skin, remove excess fish meat, cut into small pieces of 0.5cm square with scissors, put into 0.1mol / LNaOH at a ratio of 1:30 (g / mL), stir gently for 6h, change the solution every 2h, wash with distilled water until neutral, drain, remove non-collagen (4°C). Put the fish skin into 10% butanol solution at a ratio of 1:30 (g / mL), stir gently for 24h, change the solution every 8h, wash with distilled water until close to neutral, drain, remove excess fat (4°C).

[0035] The treated fish skin was then treated according to the optimized substrate concentration of 1:15, temperature of 50°C, pH 6.0, papain addition of 3500U / g, and treatment time of 5h.

[0036] The collagen solution extracted by the acid method was centrifuged in a refrigerated centrifuge (10000r / minx30min, 4°C) to obtain the supernatant, sodium chloride was added to a final concentration of 0.9M and the mixture was allowed to stand for salting out for 12h, centrifuged (10000r / min, 20min, 4°C) to obtain the precipitate, the precipitate was redissolved with 1mol sodium chloride solution of 0.05mol / L Tris-HCl (pH=7.4) for 12h, centrifuged (10000r / min, 20min, 4°C) to obtain the supernatant, and sodium chloride was used for secondary salting out. Sodium chloride was added to a final concentration of 2.5M and the mixture was allowed to stand for salting out for 12h, centrifuged (10000r / min, 20min, 4°C) to discard the supernatant, and the obtained precipitate was redissolved with 0.5M acetic acid for 12h for dialysis. The collagen precipitate obtained by salting out was soaked in 10 times deionized water at 4°C for several minutes. When the precipitate became transparent, it was centrifuged and the supernatant was sucked out. It was redissolved in 0.5M acetic acid solution and slowly stirred to dissolve. It was placed in a dialysis bag and dialyzed at 4°C. The molecular weight cutoff of the dialysis bag was 14000. The re-dissolved solution was dialyzed against 0.5M acetic acid, 0.1M acetic acid and deionized water respectively until it was dialyzed with 0.1MAgNO3. It was checked that there was no Cl in the external solution. -1 The dialysis was terminated at 1 000 ℃, and the collagen was obtained by freeze drying. The extraction rate of collagen reached 96.53%.

[0037] Example 4 Detection of Antioxidant Ability of Collagen

[0038] Determination of superoxide anion free radical scavenging ability: Take 4.5mL of 50mmol / L (pH8.2) Tris-HCl buffer in a test tube, add 2mL of distilled water, add 1mL of collagen solution of different concentrations (1mg / mL, 10mg / mL, 50mg / mL), mix thoroughly, add 1mL of distilled water to the blank group instead of the sample solution, react at 25℃ for 20min, add 0.5mL of 3mmol / L pyrogallol solution, shake quickly, measure the absorbance at 325nm wavelength (with buffer solution as reference solution), read once every 30s within 4min, and calculate the average oxidation rate within 4min. Each concentration group was measured 3 times in parallel, and the average value was taken. The measurement results were expressed as clearance rate, and the calculation formula was: Clearance rate (%) = (k0-k1) / k0×100% Wherein: k0-reaction rate of pyrogallol self-oxidation; k1-reaction rate of pyrogallol self-oxidation after adding sample solution. The results are shown in Table 2.

[0039] Table 2 Antioxidant capacity test of each group

[0040] Group Clearance rate (%) Blank control group - 1mg / mL collagen group 35.14±0.35 10mg / mL collagen group 56.48±0.64 50mg / mL collagen group 89.57±1.36

[0041] - indicates no clearance.

[0042] It can be seen from Table 2 that the scavenging ability of collagen peptides for superoxide anion free radicals increases with the increase of sample solution concentration. When the sample solution concentration is 50 mg / mL, the scavenging rate can reach (89.57±1.36)%.

[0043] Example 5: Efficacy verification of anti-aging polypeptide KS-C-6 and / or collagen

[0044] SPF KM female mice were randomly divided into 6 groups, 10 mice in each group, namely NC normal control group, SLM aging model group, KS-C-6 polypeptide group, collagen group, KS-C-6 polypeptide combined with collagen group, and positive control group; wherein, the mice in the NC control group were subcutaneously injected with an equal volume of 0.85% saline at the nape of the neck every day; the mice in the SLM group were subcutaneously injected with 300 mg / kg of D-galactose at the nape of the neck every day; the mice in the other experimental groups were subcutaneously injected with 300 mg / kg of D-galactose every day for modeling, and then the corresponding collagen or KS-C-6 polypeptide or Vc was administered orally once every morning. The specific administration forms were as follows: KS-C-6 polypeptide group was gavaged with 10 mg / kg; collagen group was gavaged with 50 mg / kg of collagen prepared in Example 3; KS-C-6 polypeptide combined with collagen group was gavaged with 50 mg / kg of collagen prepared in Example 3 and 10 mg / kg of KS-C-6 polypeptide; the positive control group was gavaged with Vc 10 mg / kg; each group was gavaged continuously for 6 weeks, the mice were weighed once a week, and the dosage volume (0.1 mL / 10 g) was calculated based on the body weight.

[0045] Each group of mice was anesthetized with 1% sodium barbital (3.5 μL / g), and 2 cm × 2 cm of dorsal skin was cut, subcutaneous tissue was cut off, and rinsed with pre-cooled saline. The skin was cut into 2 pieces, and the ELISA Kit was used to measure the hydroxyproline content in the skin tissue of one piece. The results are shown in Figure 2 shown.

[0046] Hydroxyproline is one of the unique components of skin collagen, which can promote the growth of skin fibroblasts and effectively reflect the degree of skin aging. Figure 2 The results showed that compared with the NC group, the hydroxyproline content in the SLM group was significantly reduced (P<0.05), indicating that the D-galactose aging mouse model was successfully established; and after treatment with KS-C-6 peptide and / or collagen, the hydroxyproline content in the skin was significantly increased, especially the hydroxyproline content in the KS-C-6 peptide combined with collagen group (9.81±0.38) μg / mL, which can effectively alleviate skin aging.

[0047] The skin of mice was pathologically scored according to the skin injury scoring standard of each group of mice by referring to the method of Kang Yuli et al. (The effect of Jiulong worm on the skin of aging mice). The specific steps include sampling: cutting a part of the skin tissue of the test mouse, and quickly fixing it with 4% paraformaldehyde neutral solution for 24 hours. The fixed tissue was taken out and placed in a fume hood, flattened with a scalpel, placed in a dehydration box with a corresponding label, dehydrated, waxed, embedded, sliced, and HE stained for observation. The results are shown in Table 3.

[0048] Table 3 Pathological scores of each group (points)

[0049] Group Pathological score (points) NC Group 1.51±0.07# SLM Group 3.68±0.19 KS-C-6 peptide group 2.34±0.12# Collagen group 3.01±0.16 KS-C-6 peptide combined with collagen group 1.83±0.09# Positive control group 3.12±0.18

[0050] As can be seen from Table 3, the scores of the SLM group were significantly higher than those of the NC group, the polypeptide group, and the polypeptide combined with collagen group (P<0.05), especially the pathological score of the KS-C-6 polypeptide combined with collagen group was significantly reduced, which shows that the polypeptide combined with collagen of the present invention has a significant synergistic therapeutic effect.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it; although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace part or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-aging polypeptide KS-C-6 with antioxidant properties, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the anti-aging polypeptide KS-C-6 as claimed in claim 1 in the preparation of a pharmaceutical composition for anti-aging.

3. The use according to claim 2, characterized in that The pharmaceutical composition also contains a second therapeutic agent, which is collagen.

4. The use according to claim 3, characterized in that The collagen is separated from fish skin.

5. The use according to claim 3, characterized in that The collagen is prepared by enzymatic separation from fish skin.

6. The method according to any one of claims 2 to 5, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that The pharmaceutical composition also comprises an antimicrobial agent and an excipient.

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