Collagen-containing composition and its application in anti-aging

Abstract

The present invention provides a composition containing collagen and its application in anti-aging. More specifically, it provides an anti-aging polypeptide KS-C-6, which has good antioxidant and anti-apoptotic properties. Preparing a composition with the anti-aging polypeptide KS-C-6 and collagen can effectively increase the water content of the skin and treat the anti-aging effect of the skin.

Description

Technical Field

[0001] The present application relates to the field of biology, and more specifically to compositions containing collagen and their application in anti-aging. Background Art

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

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

[0004] In addition, oxidative stress is one of the most important causes of the aging process. Ultraviolet radiation can induce the production of ROS, leading to oxidative damage and the accumulation of oxidation products, which are all markers of oxidative stress. The damage caused by ROS is one of the important mechanisms leading to skin aging. The sources of ROS in cells include enzymatic and non-enzymatic ones. Enzymes that produce reactive oxygen species include the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidoreductase (XOR), several peroxisomal oxidases, the cytochrome P450 family, cyclooxygenase, and lipoxygenase. ROS can be produced not only in mitochondria through respiration but also in the endoplasmic reticulum, cytoplasm, cell membrane, and peroxisomes. According to the free radical theory of aging, ROS mainly produced by oxidative cell metabolism play an important role in chronological aging and photoaging. ROS cause 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 skin aging process has become one of the hotspots in current life science research. Currently, collagen has been widely used in delaying skin aging, and there are many records of collagen products such as tortoise shell glue, donkey-hide gelatin, deer horn glue, turtle shell glue, and fish glue in traditional Chinese medicine classics represented by Compendium of Materia Medica. Collagen products from sources such as salmon, jellyfish, and sea cucumbers have the effect of improving skin quality. The potential ability of collagen to delay skin aging has also been reported abroad. Generally speaking, the application of collagen in delaying skin aging is very extensive.

[0006] Specifically, studies have shown that collagen polypeptides extracted from cod skin have good moisture absorption and retention properties at different relative humidities, can reduce the damage caused by ultraviolet radiation, and also have multiple effects such as anti-aging and repairing damaged skin, showing great potential in skin care product moisturization. In addition, palmitoyl peptide repairs the aging damage of the skin through the adsorption and interaction with keratin in the stratum corneum and hair cuticle, and the innovative cosmeceutical ointment added with this peptide is safe and non-irritating. In vivo studies on oyster protein hydrolysate polypeptides with a molecular weight range of 302.17 - 2936.43 Da have shown that topical application on the skin can significantly reduce water loss, epidermal hyperplasia, and the degradation of collagen and elastic fiber caused by chronic ultraviolet B irradiation, and has good moisturizing and anti-wrinkle effects while protecting the skin from photo-damage. Chitosan / collagen peptide nanoparticles with biocompatibility and biodegradation gradients stabilizing Pickering emulsions have been rated as effective green biodegradable topical carriers, and the moisturizing property of the emulsion can be affected by changing the ratio of the oil phase and the water phase, which is more targeted for people with different skin types. Studies have shown that sea cucumber collagen peptides were compared with tilapia and pig skin collagen peptides, and the commercial value of sea cucumber industrial by-products is higher, but all three types of collagen peptides showed better moisture absorption and retention abilities than glycerol. Generally speaking, the moisturizing effects of collagen polypeptides from different sources and molecular weight distributions are slightly different. Compared with the moisture absorption, retention, and antioxidant activities of collagen peptides from pig skin, cowhide, cod skin, and bovine bone, donkey skin collagen peptides perform better. In addition, some synthetic collagen polypeptides have also been proven to have specific skin care moisturizing and anti-aging effects. For example, an innovative neuropeptide - acetyl hexapeptide-8 is used in cosmetic formulations, which can not only enhance skin moisturization but also reduce wrinkles caused by facial muscle contraction. Moreover, palmitoyl pentapeptide-3 obtained by compounding natural collagen polypeptides with palmitic acid can significantly improve skin affinity and water retention, and effectively promote the regeneration of skin collagen, hyaluronic acid, and elastic fibers, thereby reducing skin fine lines and greatly enhancing skin firmness and gloss.

[0007] Although there are currently various treatment forms for skin aging, compositions with good and persistent treatment effects still need to be further developed and prepared, and the market demand is strong. Summary of the Invention

[0008] The present invention screens and isolates an anti-aging polypeptide KS-C-6 with good antioxidant properties from cuttlefish skin, and its amino acid sequence is shown in SEQ ID NO: 1.

[0009] Furthermore, the composition prepared from the anti-aging polypeptide KS-C-6 and collagen of the present invention 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 and collagen of the present invention.

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

[0012] More specifically, the preparation method of the collagen is as follows: Take fresh cuttlefish skin, remove the excess fish meat, cut it into small squares with a side length of 0.5 cm with scissors, put it into 0.1 mol / L NaOH according to the ratio of 1:30 (g / mL), stir gently for 6 h, change the solution every 2 h, then wash it with distilled water until neutral, drain, and remove non-collagen. Then put the fish skin into a 10% butanol solution according to the ratio of 1:30 (g / mL), stir gently for 24 h, change the solution every 8 h, then wash it with distilled water until nearly neutral, drain, and remove the excess fat. Subsequently, the treated fish skin is processed according to the optimized substrate concentration of 1:15, temperature of 50 °C, pH 6.0, the enzyme addition amount of papain is 3500 U / g, and the treatment time is 5 h. The collagen solution extracted by the acid method is centrifuged with a refrigerated centrifuge to take the supernatant, sodium chloride is added and allowed to stand for salting out, the precipitate is centrifuged to take, the precipitate is redissolved in a 1 mol sodium chloride solution of 0.05 mol / L Tris-HCI (pH = 7.4) for 12 h, then centrifuged to take the supernatant, and secondary salting out is continued with sodium chloride, the supernatant is centrifuged and discarded, and the obtained precipitate is redissolved in 0.5 M acetic acid for 12 h and waited for dialysis. The collagen precipitate obtained by salting out is soaked in 10 times deionized water at 4 °C for several minutes. When the precipitate becomes transparent, centrifuge and suck out the supernatant, redissolve it in 0.5 M acetic acid solution, stir slowly to dissolve it, put it into a dialysis bag and dialyze at 4 °C. The molecular weight cut-off of the dialysis bag is 14000, and the reconstituted solution is dialyzed against 0.5 M acetic acid, 0.1 M acetic acid, and deionized water respectively until no Cl -1 is detected in the external solution with 0.1 M AgNO3, and dialysis is terminated, and then freeze-dried to obtain collagen.

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

[0014] There is also provided a pharmaceutical composition comprising the polypeptide of the present invention. Such a composition comprises a therapeutically or prophylactically effective amount of the polypeptide or protein admixed with pharmaceutically acceptable materials and physiologically acceptable formulation materials. The pharmaceutical composition may comprise formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or permeability of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents; flavoring agents and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as 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 (such as alkali metal halides (preferably sodium chloride or potassium, mannitol, sorbitol)); delivery vehicles; diluents; excipients.

[0015] The nature of the main vehicle or carrier in a pharmaceutical composition can be aqueous or non-aqueous. For example, suitable vehicles or carriers can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions contain a Tris buffer at about pH 7.0 - 8.5, or an acetate buffer at about pH 4.0 - 5.5, which may further include sorbitol or a suitable alternative thereof. In one embodiment of the present invention, the composition can be prepared for storage in the form of a lyophilized mass or an aqueous solution by mixing the selected composition of desired purity with optional formulation reagents. Additionally, 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 mixture of granules (optionally, after grinding) to obtain a tablet or lozenge core. Suitable auxiliaries can be added if needed. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol and sorbitol; starches from corn, wheat, rice, potato or other plants; celluloses, such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; gums, including gum arabic and tragacanth; and proteins such as gelatin and collagen. Disintegrants or solubilizers, such as cross-linked polyvinylpyrrolidone, agar and alginic acid or its salts, such as sodium alginate, can be added if needed.

[0017] The route of administration of the pharmaceutical composition follows known methods, such as oral, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous or intraperitoneal injection; and intranasal, enteral, topical, sublingual, urethral, vaginal or rectal methods, administered by sustained release systems or by implant devices. If needed, the composition can be administered by bolus or continuous infusion or by implant device. Alternatively or additionally, the composition can be administered locally via an implanted membrane, sponge or other suitable material that has adsorbed or encapsulated the desired molecule. When using an implant device, the device can be implanted into any suitable tissue or organ, and the delivery of the desired molecule can be via diffusion, timed release pellets or continuous administration.

[0018] The effective amount of the pharmaceutical composition employed in treatment will depend, for example, on the treatment context and purpose. Those skilled in the art will understand that the appropriate dosage levels for treatment will thus vary in part depending on the molecule being delivered, the indication for which the polypeptide is used, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, the clinician can titrate the dose and modify the route of administration to achieve optimal efficacy. General dosages can be from 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 administered by intravenous injection or infusion. 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 particular formulation. The dosing frequency will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Generally, the composition is administered until a dose is reached that achieves the desired effect. The composition can thus be administered as a single dose or multiple doses (at the same or different concentrations / dose) over time, or as a continuous infusion. Further refinement of the appropriate dose is routinely carried out. The appropriate dose can be determined by using appropriate dose-response data.

[0019] Beneficial effects

[0020] The present invention provides a composition comprising collagen and its application in anti-aging. More specifically, it provides the anti-aging polypeptide KS-C-6, which has good antioxidant and anti-apoptotic properties. Preparing the anti-aging polypeptide KS-C-6 and collagen together into a composition can effectively increase the water content of the skin and treat the anti-aging effect of the skin. Description of the drawings

[0021] Figure 1 Graph of the identification results of the antioxidant properties of the anti-aging polypeptide KS-C-6

[0022] Figure 2 Graph of the results of the effects of each group on the content of hydroxyproline in skin tissue Detailed implementation manners

[0023] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions 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 through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement 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 polypeptide KS-C-6

[0025] Previously, the applicant screened and obtained the polypeptide KS-C-6 from cuttlefish skin, and its amino acid sequence is shown in SEQ ID NO: 1, and entrusted Shanghai Qiangyao Biotechnology for synthesis. HFF-1 human skin fibroblasts (Zhongqiao Xinzhou, product number ZQ0450) were cultured in DMEM 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%, subculture was carried out, and subculture was carried out every 2 days.

[0026] Select HFF-1 cells in the logarithmic growth phase, digest with 0.25% trypsin, and adjust the cell concentration to 5×10 4 cells / mL. 100 μL of cell suspension was inoculated into each well of a 96-well culture plate and cultured in an incubator at 37 °C and 5% CO2 for 24 h. Observed under an inverted microscope, the cells were completely adherent and in good growth condition. Carefully aspirate the old medium with a 1 mL syringe. In the sample group, add the medium with an optimized concentration of 120 μmol / L H2O2. After culturing for 24 h, aspirate the medium and add the medium containing three concentrations of polypeptide (the concentrations of polypeptide KS-C-6 are 50 μg / mL, 100 μg / mL, and 200 μg / mL respectively, and the positive control is 200 μg / mL Vc). The blank control group was added with blank medium. After culturing in an incubator at 37 °C and 5% CO2 for 24 h, the cell proliferation rate was measured by the CCK-8 method. The cell proliferation rate = (A1 - A2) / (A3 - A2)×100%, where A1 represents the absorbance of the well containing cells, CCK-8 solution and different concentrations of drugs; A2 represents the absorbance of the well containing CCK-8 solution, no cells, and no drugs; A3 represents the absorbance of the well containing cells and CCK-8 solution but no samples.

[0027] The protective ability of the polypeptide against oxidative damage of fibroblasts is shown in Figure 1 As can be seen from Figure 1 , 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 condition of a concentration of 200 μg / mL, the cell proliferation rate (119.13 ± 2.03)% produced by the polypeptide of the present invention is higher than that of the positive control, indicating that the polypeptide of the present invention has a better promotion effect.

[0028] Example 2 Detection of the apoptosis rate of cells

[0029] Select HFF-1 cells in the logarithmic growth phase, digest with 0.25% trypsin, and adjust the cell suspension concentration to 8×10 4Cells were inoculated at a density of cells / mL into a 6-well culture plate at 1 mL per well and cultured in an incubator at 37 °C with 5% CO2 for 24 h. Observation under an inverted microscope showed that the cells had completely adhered and were in good growth condition. The old culture medium was carefully aspirated with a 1 mL syringe, and culture media containing (the concentrations of polypeptide KS-C-6 were 50 μg / mL, 100 μg / mL, and 200 μg / mL respectively, and the positive control was 100 μg / mL Vc) were added respectively and cultured for 24 h. The culture medium without samples was used as the blank control. The cells were pretreated with an AnnexinV-PE / 7-AAD apoptosis kit and detected using a flow cytometer. The parallel experiment was repeated 3 times. The results are shown in Table 1.

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

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

[0032] As can be seen from Table 1, compared with the blank control, both the polypeptide and the positive control could significantly reduce the apoptosis rate of skin fibroblasts. After the polypeptide acted at 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] Fresh hairtail skin was taken, and the excess fish meat was removed. It was cut into small squares with a side length of 0.5 cm with scissors and placed in 0.1 mol / L NaOH at a ratio of 1:30 (g / mL). It was gently stirred for 6 h, and the solution was changed every 2 h. Then it was washed with distilled water until neutral, drained, and non-collagen was removed (at 4 °C). Then the fish skin was placed in a 10% butanol solution at a ratio of 1:30 (g / mL), gently stirred for 24 h, and the solution was changed every 8 h. Then it was washed with distilled water until nearly neutral, drained, and excess fat was removed (at 4 °C).

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

[0036] The collagen solution extracted by the acid method was centrifuged with a refrigerated centrifuge (10000 r / min × 30 min, 4°C), and the supernatant was taken. Sodium chloride was added to a final concentration of 0.9 M, and the mixture was allowed to stand for salting out for 12 h. Then it was centrifuged (10000 r / min, 20 min, 4°C), and the precipitate was taken. The precipitate was redissolved in a 1 mol sodium chloride solution of 0.05 mol / L Tris-HCl (pH = 7.4) for 12 h, and then centrifuged (10000 r / min, 20 min, 4°C) to take the supernatant. Secondary salting out was continued with sodium chloride, and sodium chloride was added to a final concentration of 2.5 M and allowed to stand for salting out for 12 h. Then it was centrifuged (10000 r / min, 20 min, 4°C), and the supernatant was discarded. The obtained precipitate was redissolved in 0.5 M acetic acid for 12 h and awaited 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 completely aspirated, redissolved in 0.5 M acetic acid solution, and slowly stirred to dissolve it. It was filled into a dialysis bag and dialyzed at 4°C. The molecular weight cut-off of the dialysis bag was 14000. The reconstituted solution was dialyzed against 0.5 M acetic acid, 0.1 M acetic acid, and deionized water respectively until no Cl -1 was detected in the external solution with 0.1 M AgNO3, and then freeze-dried to obtain collagen. After detection, the extraction rate of collagen reached 96.53%.

[0037] Example 4 Detection of the antioxidant capacity of collagen

[0038] Determination of the scavenging ability against superoxide anion radicals: Take 4.5 mL of 50 mmol / L (pH 8.2) Tris-HCl buffer solution in a test tube, add 2 mL of distilled water, add 1 mL of collagen solutions with different concentrations (1 mg / mL, 10 mg / mL, 50 mg / mL), and mix well. In the blank group, 1 mL of distilled water was added instead of the sample solution. After reacting at a constant temperature of 25°C for 20 min, 0.5 mL of 3 mmol / L pyrogallol solution was added, and it was quickly shaken well. The absorbance was measured at a wavelength of 325 nm (using the buffer solution as the reference solution). Within 4 min, readings were taken every 30 s, and the average oxidation rate within 4 min was calculated. Each concentration group was measured in parallel 3 times, and the average value was taken. The measurement results were expressed as the scavenging rate. The calculation formula was: Scavenging rate (%) = (k0 - k1) / k0 × 100% where: k0—the reaction rate during the autoxidation of pyrogallol; k1—the reaction rate during the autoxidation of pyrogallol after adding the sample solution. The results are shown in Table 2.

[0039] Table 2 Detection of the antioxidant capacity of each group

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

[0041] - indicates no scavenging rate.

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

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

[0044] SPF-grade female KM mice were randomly divided into 6 groups, with 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. Among them, the mice in the NC control group were subcutaneously injected with an equal volume of 0.85% normal 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. After the other experimental groups were subcutaneously injected with 300 mg / kg of D-galactose to establish the model, they were intragastrically administered the corresponding collagen or KS-C-6 polypeptide or Vc once a day by mouth in the morning. The specific administration forms were as follows: the KS-C-6 polypeptide group was intragastrically administered 10 mg / kg; the collagen group was intragastrically administered 50 mg / kg of the collagen prepared in Example 3; the KS-C-6 polypeptide combined with collagen group was intragastrically administered 50 mg / kg of the collagen prepared in Example 3 and 10 mg / kg of KS-C-6 polypeptide; the positive control group was intragastrically administered 10 mg / kg of Vc; each group was continuously intragastrically administered for 6 weeks, and the body weight of the mice was weighed once a week, and the administration volume (0.1 mL / 10 g) was calculated according to the body weight.

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

[0046] Hydroxyproline is one of the specific components that make up skin collagen, which can promote the growth of skin fibroblasts and effectively reflect the degree of skin aging. From Figure 2 the results, it can be seen that compared with the NC group, the content of hydroxyproline in the SLM group was significantly decreased (P<0.05), indicating that the D-galactose-induced aging mouse model was successfully established. After treatment with KS-C-6 polypeptide and / or collagen, the content of hydroxyproline in the skin was significantly increased. Especially, the content of hydroxyproline in the KS-C-6 polypeptide combined with collagen group was (9.81±0.38) μg / mL, which could effectively relieve skin aging.

[0047] The skin of each group of mice was pathologically scored according to the skin injury scoring criteria of Kang Yuli et al. (The effect of Tenebrio molitor on the skin of senile mice). The specific steps included specimen collection: a part of the skin tissue of the test mice was excised and quickly fixed in 4% neutral paraformaldehyde solution for 24 hours. The fixed tissue was taken out and placed in a fume hood, trimmed flat with a scalpel, placed in a corresponding labeled dehydration box, dehydrated, infiltrated with wax, embedded, sectioned, and observed by HE staining. 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 polypeptide group 2.34±0.12# Collagen group 3.01±0.16 KS-C-6 polypeptide combined with collagen group 1.83±0.09# Positive control group 3.12±0.18

[0050] As can be seen from Table 3, the score of the SLM group was significantly different from that of the NC group, the polypeptide group, and the polypeptide combined with collagen group (P<0.05). In particular, the pathological score of the KS-C-6 polypeptide combined with collagen group was significantly reduced, indicating that the polypeptide combined with collagen of the present invention has a significant synergistic therapeutic effect.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, 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 solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; 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 as shown in SEQ ID NO:

1.

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

3. The use according to claim 2, characterized in that A second therapeutic agent is further added to the pharmaceutical composition, and the second therapeutic agent is collagen.

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

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

6. The use according to any one of claims 2-5, 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 further comprises an antimicrobial agent and an excipient.

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