Biological preparation for improving collagen expression of skin cells

Abstract

The invention provides a biological preparation for improving collagen expression of skin cells, and belongs to the technical field of skin medicine. The biological agent is prepared from grass carp roe polypeptide and water. The grass carp roe polypeptide with the molecular weight smaller than 1 kDa is obtained through liquid nitrogen freezing and smashing, supercritical CO2 degreasing, alkaline condition regulation and control and ultrasonic-assisted extraction. Experimental results show that the grass carp roe polypeptide prepared by the invention significantly promotes collagen gene expression, improves the hydroxyproline level in skin tissues in a D-galactose induced aging mouse model, and effectively slows down skin aging. The grass carp roe polypeptide provided by the invention provides a new bioactive component for the fields of beauty and skin care, medical dressings and the like, and meanwhile, high value-added utilization of grass carp resources is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of skin medicine, and in particular relates to a biological preparation for improving the expression of collagen in skin cells. Background Art

[0002] Collagen is the most abundant structural protein in mammals, accounting for about 30% of the total body protein. In the skin, collagen is the main component of the dermis, accounting for about 70%-80% of the dry weight of the skin. It is composed of three polypeptide chains (α chains) spirally wound to form a stable triple helix structure, which gives the skin strength and elasticity. Collagen not only plays a key role in maintaining the structural integrity of the skin, but also participates in wound healing and tissue repair.

[0003] There are many types of collagen, among which type I and type III collagen are the most common in the skin. Type I collagen mainly provides mechanical strength to the skin, while type III collagen gives the skin elasticity and flexibility. The synthesis of collagen is mainly completed by fibroblasts in the skin, and the synthesis process includes the translation, secretion, shearing and assembly of procollagen. However, with age, collagen in the skin is gradually lost, the synthesis rate slows down, and the degradation rate accelerates. This process leads to aging phenomena such as decreased skin elasticity, increased wrinkles and sagging. Therefore, promoting collagen expression in skin cells is the key to effectively reducing skin aging and decreased elasticity.

[0004] Grass carp is an important freshwater economic fish with a wide range of utilization potential due to its abundant resources and low price. However, grass carp roe is often neglected or discarded during aquatic processing and has not been effectively utilized. Therefore, using peptides extracted from grass carp roe to promote collagen expression in skin cells can not only increase the added value of grass carp, but also provide a new source of raw materials for the development of beauty and skin care products. Summary of the invention

[0005] The purpose of the present invention is to provide a biological preparation for increasing the expression of collagen in skin cells, thereby effectively slowing down the aging rate of skin cells.

[0006] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0007] A biological preparation for improving the expression of collagen in skin cells. The biological preparation is a liquid biological preparation and is composed of grass carp egg polypeptide and water.

[0008] Furthermore, the grass carp egg polypeptide is prepared by the following preparation method:

[0009] Clean the fresh grass carp roe and refrigerate at 4°C;

[0010] Refrigerated grass carp roe were quickly frozen in liquid nitrogen and then ground into powder;

[0011] Degreasing the powder with supercritical CO2, and leaving the powder to stand until the CO2 is completely released;

[0012] Distilled water was added at a solid-liquid ratio of 1:20, the pH value was adjusted to 8, and ultrasonic treatment was performed at 180 W ultrasonic power for 60 min;

[0013] The supernatant was collected by centrifugation, the pH of the supernatant was adjusted to 3, and after standing at room temperature, the protein precipitate was collected by centrifugation;

[0014] The protein precipitate was dissolved in distilled water to prepare a protein solution, and the pH value of the protein solution was adjusted to 7.5;

[0015] Add flavor protease at an enzyme dosage of 4000U / g protein precipitate and perform enzymolysis at 50°C for 4 hours;

[0016] The enzymatic hydrolysate is heated to terminate the enzyme activity, and the supernatant is collected by centrifugation;

[0017] The supernatant was treated by an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the retentate a and the filtrate a were collected;

[0018] The filtrate was processed through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the retentate b and the filtrate b were collected;

[0019] The filtrate b is placed in a freeze dryer for vacuum freeze drying to obtain grass carp egg polypeptide.

[0020] Furthermore, in the biological preparation, the concentration of the grass carp egg polypeptide is 25 μg / mL-300 μg / mL.

[0021] Further, the skin cells are human skin fibroblasts;

[0022] The collagen is type I collagen and type III collagen.

[0023] Furthermore, in the biological preparation, the concentration of grass carp egg polypeptide is 150 μg / mL.

[0024] A use of a grass carp egg polypeptide in the preparation of a biological preparation for improving the expression of collagen in skin cells. The grass carp egg polypeptide is prepared according to the above-mentioned grass carp egg polypeptide preparation method.

[0025] Furthermore, the skin cells are human skin fibroblasts, and the collagen is type I collagen and type III collagen;

[0026] In the biological preparation, the concentration of grass carp egg polypeptide is 25 μg / mL-300 μg / mL.

[0027] Furthermore, in the biological preparation, the concentration of grass carp egg polypeptide is 150 μg / mL.

[0028] A use of a grass carp egg polypeptide in the preparation of a biological preparation for slowing down skin aging. The grass carp egg polypeptide is prepared according to the above-mentioned grass carp egg polypeptide preparation method.

[0029] Furthermore, the biological agent for slowing down skin aging achieves the effect of slowing down skin aging by promoting the expression of type I collagen and type III collagen;

[0030] The skin aging is skin aging caused by D-galactose;

[0031] In the biological preparation, the concentration of the grass carp egg polypeptide is 25 μg / mL-300 μg / mL.

[0032] Furthermore, in the biological preparation, the concentration of grass carp egg polypeptide is 150 μg / mL.

[0033] The invention discloses an application of a grass carp egg polypeptide in the preparation of a biological agent for improving the growth of skin cells, characterized in that the grass carp egg polypeptide is prepared according to the above-mentioned grass carp egg polypeptide preparation method.

[0034] Further, the skin cells are human skin fibroblasts;

[0035] In the biological preparation, the concentration of the grass carp egg polypeptide is 150-300 μg / mL.

[0036] Furthermore, in the biological preparation, the concentration of grass carp egg polypeptide is 150 μg / mL.

[0037] The beneficial effects of the present invention are:

[0038] The present invention provides a method for preparing a grass carp egg polypeptide that can significantly promote the expression of collagen in skin cells. Through unique process optimization, including liquid nitrogen freezing and crushing, supercritical CO2 degreasing, ultrasound-assisted extraction under alkaline conditions, and two-stage ultrafiltration, a grass carp egg polypeptide that can significantly promote the expression of collagen genes in skin fibroblasts is successfully obtained. The polypeptide can show certain biological activity at low concentrations and significantly promote the gene expression of type I and type III collagen.

[0039] In the D-galactose-induced mouse skin aging model, grass carp egg polypeptide c showed significant anti-aging effects. The polypeptide significantly increased the hydroxyproline content in skin tissue at a concentration of 150 μg / mL, inhibited the degradation of collagen, and significantly delayed the skin aging phenomenon caused by D-galactose. In addition, the polypeptide can significantly promote the proliferation of skin fibroblasts at high concentrations, providing additional support for skin repair.

[0040] Secondly, the grass carp roe polypeptide of the present invention not only shows a wide range of application prospects in the development of biological preparations such as beauty and skin care products and medical dressings, but also realizes the high added value utilization of grass carp roe, a low-cost resource, reduces the waste of aquatic resources, and enhances the value of the aquatic economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The effects of grass carp egg peptides obtained by different preparation methods on the expression of type I collagen gene (COL1A1) are shown;

[0042] Figure 2 The effects of grass carp egg peptides obtained by different preparation methods on the expression of type III collagen gene (COL3A1) are shown;

[0043] Figure 3 The effect of grass carp egg polypeptide c on COL1A1 gene expression at different concentrations (5 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 150 μg / mL, 300 μg / mL) is shown. DETAILED DESCRIPTION

[0044] Several embodiments of the present invention will be described in detail below. These details are intended to further illustrate specific aspects, features and implementation methods of the present invention, rather than to restrict the invention. It should be appreciated that the terms used in the present invention are intended to describe specific implementation methods, rather than to limit the present invention.

[0045] Example 1

[0046] Fresh grass carp roe were repeatedly washed with cold distilled water to remove blood, mucus and impurities on the surface of the roe, and the cleaned roe were refrigerated and stored at 4°C to prevent protein degradation;

[0047] The refrigerated fish eggs are quickly frozen in liquid nitrogen and then ground into powder using a cryogenic pulverizer;

[0048] The powder was placed in a supercritical carbon dioxide extraction device for supercritical CO2 degreasing at a temperature of 35°C and a pressure of 25 MPa for 1.5 hours. After degreasing, the powder was allowed to stand for 30 minutes until the CO2 was completely released.

[0049] Weigh 10 g of grass carp roe powder and add 200 ml of distilled water at a solid-liquid ratio of 1:20;

[0050] The pH value was adjusted to 8 using 1 mol / L NaOH solution, and ultrasonic treatment was performed at 180 W ultrasonic power for 60 min;

[0051] Centrifuge at 8000 rpm for 15 minutes, collect the supernatant, adjust the pH of the supernatant to 3 with 1 mol / L HCl solution, let stand at room temperature for 30 minutes, and centrifuge at 8000 rpm for 10 minutes to collect the protein precipitate;

[0052] The protein precipitate was dissolved in distilled water to prepare a protein solution with a mass concentration of 2%, and the pH value of the solution was adjusted to 7.5 using 1 mol / L NaOH solution;

[0053] Add flavor protease at an enzyme dosage of 4000U / g protein precipitate and perform enzymolysis at 50°C for 4 hours;

[0054] Heat the enzymatic solution to 95°C for 10 minutes to terminate the enzyme activity;

[0055] Centrifuge at 8000 rpm for 15 min and collect the supernatant;

[0056] The supernatant was treated by an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the retentate a (>5 kDa) and the filtrate a (<5 kDa) were collected;

[0057] The filtrate was processed through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the retentate b (1-5 kDa) and the filtrate b (<1 kDa) were collected;

[0058] The retentate a, the retentate b and the filtrate b are placed in a freeze dryer for vacuum freeze drying to obtain grass carp egg polypeptides a, b and c.

[0059] Example 2

[0060] Fresh grass carp roe was cleaned and impurities were removed, the roe was mixed with an equal volume of cold distilled water, and homogenized at high speed (10000 rpm for 2 minutes) using a homogenizer to obtain a roe homogenate;

[0061] Centrifuge at 8000 r / min at 4°C for 15 min, and collect the supernatant;

[0062] The pH value of the supernatant was adjusted to 9, and alkaline protease was added at an enzyme dosage of 4000 U / g, and enzymolysis was performed at 50°C for 3 hours;

[0063] After the enzymatic hydrolysis was completed, the enzymatic solution was heated to 100°C and maintained for 10 minutes to terminate the enzyme activity;

[0064] Centrifuge at 8000 rpm for 15 min and collect the supernatant;

[0065] The supernatant was treated by an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the retentate A (>5 kDa) and the filtrate A (<5 kDa) were collected;

[0066] The filtrate was processed through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the retentate B (1-5 kDa) and the filtrate B (<1 kDa) were collected;

[0067] The retentate A, the retentate B and the filtrate B are placed in a freeze dryer for vacuum freeze drying to obtain grass carp egg polypeptides A, B and C.

[0068] Example 3

[0069] Clean fresh grass carp roe, drain the water, and place the roe in a high pressure steam sterilizer at 121°C for 15 minutes to kill microorganisms and inactivate endogenous enzymes;

[0070] Add plant lactic acid bacteria suspension (1×10 8 CFU / mL), after being fully stirred, sealed and fermented for 72 hours to obtain a fermentation mixture;

[0071] Heat the fermentation mixture in a water bath at 80°C for 10 minutes to terminate the bacterial activity and the fermentation process;

[0072] Centrifuge at 4°C, 10,000 rpm for 20 minutes, collect the supernatant, adjust the pH to 7, add neutral protease at an enzyme dosage of 4,000 U / g, and perform enzymolysis at 45°C for 3 hours;

[0073] Heat the enzymatic solution to 95°C for 10 minutes to terminate the enzyme activity;

[0074] Centrifuge at 8000 rpm for 15 min and collect the supernatant;

[0075] The supernatant was processed through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the retentate 1 (>5 kDa) and the filtrate 1 (<5 kDa) were collected;

[0076] The filtrate was processed through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the retentate 2 (1-5 kDa) and the filtrate 2 (<1 kDa) were collected;

[0077] The retentate 1, the retentate 2 and the filtrate 2 are placed in a freeze dryer for vacuum freeze drying to obtain grass carp egg polypeptides I, II and III.

[0078] Example 4

[0079] Comparison of the effects of peptides obtained by different preparation methods on the expression of type I collagen gene COL1A1

[0080] The culture medium of human skin fibroblasts cultured to the logarithmic growth phase was discarded, the cells were washed once with PBS, and an appropriate amount of trypsin-EDTA solution was added and digested at 37°C for about 2 minutes until the cells became round and loose;

[0081] Add medium containing FBS to terminate digestion, gently pipette the cells to suspend them, and adjust the cell concentration to 5×10 4 cells / mL, the cells were seeded into 6-well plates, and 2 mL was added to each well;

[0082] Place in an incubator (37°C, 5% CO2) and culture until the cell density reaches more than 80%;

[0083] The grass carp egg polypeptides a, b and c, grass carp egg polypeptides A, B and C, and grass carp egg polypeptides I, II and III prepared in Examples 1-3 were all prepared with a culture medium to prepare a 50 μg / mL working solution (serum-free);

[0084] The control group was replaced with 2 mL of fresh culture medium (serum-free), and the experimental groups were added with the above-configured peptide working solution (50 μg / mL, serum-free) to ensure that the experimental conditions of each group were consistent. Each 6-well plate was placed in a cell culture incubator and cultured for 48 h.

[0085] After treatment, the culture medium in the 6-well plate was discarded, and the cells were gently washed twice with pre-cooled PBS buffer to remove residual peptides and culture medium components;

[0086] Add 1 mL of TRIzol reagent to each well to evenly cover the cell surface, and pipette repeatedly to ensure that the cells are fully lysed to obtain a uniform cell lysate;

[0087] RNA was extracted according to the steps of TRIzol RNA extraction, and after passing the quality test, it was used for subsequent processing;

[0088] Reverse transcribe RNA into cDNA using a cDNA synthesis kit;

[0089] According to the following reaction system: SYBR Green qPCR Master Mix: 10 μL, forward primer (10 μM): 0.4 μL, reverse primer (10 μM): 0.4 μL, cDNA template: 1 μL, nuclease-free water: 8.2 μL;

[0090] The reaction conditions were as follows: pre-denaturation: 95°C, 5 min; cycling (40 cycles): 95°C, 10 s (denaturation), 60°C, 30 s (annealing / extension);

[0091] COL1A1 Forward Primer: 5'-catgaccgagacgtgtggaa-3';

[0092] COL1A1 Reverse Primer: 5'-ggcagttcttggtctcgtca-3';

[0093] The expression difference of gene COL1A1 after treatment with different polypeptides was detected (GAPDH was selected as the internal reference gene), and each group of experiments was repeated 3 times.

[0094] The experimental results of this embodiment are as follows Figure 1 As shown in the figure, it can be clearly seen that except for grass carp egg polypeptide A which did not reach statistical significance in promoting the relative expression level of COL1A1 gene, the other prepared grass carp egg polypeptides all showed significant promoting effects.

[0095] Through further analysis of the data, it was found that grass carp egg polypeptide c, grass carp egg polypeptide C and grass carp egg polypeptide III with a molecular weight of less than 1kDa showed relatively good effects in promoting COL1A1 gene expression. These small molecule polypeptides significantly increased the expression level of the COL1A1 gene, probably because they have good cell permeability and can more effectively bind to the receptors of skin fibroblasts, thereby activating related signaling pathways and promoting collagen synthesis.

[0096] However, although grass carp egg polypeptide C, grass carp egg polypeptide C and grass carp egg polypeptide III all showed good promoting effects, there were still some differences between their effects. This difference may be closely related to its preparation method and polypeptide structure characteristics, indicating that different extraction and treatment methods have a significant effect on the biological activity of grass carp egg polypeptides.

[0097] Example 5

[0098] In Example 4, the present invention screened out grass carp egg polypeptide c, grass carp egg polypeptide C and grass carp egg polypeptide III, which had a good promoting effect on the relative expression of gene COL1A1. Therefore, in this example, the present invention detected the effects of the above three polypeptides on the expression of type III collagen gene COL3A1.

[0099] The detection method is similar to that of Example 4, except that the gene target is COL3A1. The primer sequence of COL3A1 is as follows:

[0100] COL3A1 Forward Primer: 5'-ctcagtggagaacgtggtcc-3';

[0101] COL3A1 Reverse Primer: 5'-gatccatctcggcctggaag-3'.

[0102] The results obtained in this example are as follows Figure 2 As shown, from Figure 2 The results show that compared with the control group grass carp egg polypeptide C, grass carp egg polypeptide C and grass carp egg polypeptide III also have a better promoting effect on the expression of type III collagen gene COL3A1.

[0103] However, unlike the promotion effect of type I collagen gene COL1A1, the promotion effect of grass carp egg polypeptide III on type III collagen gene COL3A1 is relatively weak, showing a moderate effect. Among the three polypeptides, only grass carp egg polypeptide C has the most significant effect on the expression of type III collagen gene COL3A1.

[0104] This indicates that only the grass carp egg polypeptide c prepared according to the preparation method of Example 1 can significantly promote the expression of type I collagen gene COL1A1 and type III collagen gene COL3A1 in skin cells at the same time.

[0105] Example 6

[0106] Through the above experiments, the present invention found that only grass carp egg polypeptide c has the best effect on promoting the expression of collagen genes COL1A1 and COL3A1. Therefore, in this embodiment, the present invention will detect the optimal concentration of grass carp egg polypeptide c for promoting collagen (since the effects of grass carp egg polypeptide c on COL1A1 and COL3A1 are basically similar, the present invention only selects COL1A1 for detection).

[0107] The detection method was the same as that in Example 4. Grass carp egg polypeptide c was prepared into 5, 25, 50, 75, 150, and 300 μg / mL serum-free culture medium working solutions for treating cells.

[0108] The test results are as follows Figure 3 As shown, from Figure 3 The results show that compared with the control group, only when the concentration of grass carp egg polypeptide c was 5μg / mL, there was no statistically significant difference in the expression of COL1A1 gene. When the concentration of grass carp egg polypeptide c was greater than or equal to 25μg / mL, it could produce a significant promoting effect on the expression of COL1A1 gene.

[0109] Further analysis shows that when the concentration is low, the promotion effect increases significantly with the increase of concentration. When the concentration is high, the increase of the promotion effect gradually decreases. When the concentration is 150μg / mL, the best effect is basically achieved. At this time, the effect of continuing to increase the concentration of grass carp egg polypeptide c is not significant. Therefore, from the perspective of maximizing the effect, 150μg / mL is considered to be the best concentration for grass carp egg polypeptide c.

[0110] Example 7

[0111] This example detects the effect of grass carp egg polypeptide c on the proliferation of human skin fibroblasts

[0112] The culture medium of human skin fibroblasts cultured to the logarithmic growth phase was discarded, the cells were washed once with PBS, and an appropriate amount of trypsin-EDTA solution was added and digested at 37°C for about 2 minutes until the cells became round and loose;

[0113] Add medium containing FBS to terminate digestion, gently pipette the cells to suspend them, and adjust the cell concentration to 5×10 4 cells / mL, the cells were seeded into a 96-well plate, and 100 μL was added to each well;

[0114] Place the 96-well plate in a cell culture incubator and culture until the cells are completely attached;

[0115] The control group was added with complete medium containing 10% FBS, the low-concentration group was added with 25 μg / mL grass carp egg polypeptide c, and the high-concentration group was added with 150 μg / mL grass carp egg polypeptide c;

[0116] The 96-well plate was placed in a cell culture incubator and cultured for 48 h. The absorbance of each treatment group was measured using CCK-8. The experiment was repeated 3 times for each group.

[0117] The results obtained in this embodiment are shown in Table 1:

[0118] Table 1 Effects of grass carp egg polypeptide c at different concentrations on the proliferation of human skin fibroblasts

[0119] Processing Groups Absorbance (450nm) P-value Control group 0.742±0.019 Low concentration group 0.786±0.024 0.0678(P＞0.05) High concentration group 0.885±0.025 0.0014(P＜0.01)

[0120] The following conclusions can be drawn from the experimental results in Table 1: In the low concentration group (25 μg / mL), although the absorbance value was slightly higher than that of the control group, statistical analysis showed that the P value was greater than 0.05, indicating that grass carp egg polypeptide at this concentration did not significantly promote the proliferation of human skin fibroblasts.

[0121] On the contrary, in the high concentration group (150 μg / mL), the absorbance was significantly higher than that of the control group, and the P value was less than 0.01, indicating that 150 μg / mL grass carp egg polypeptide c can promote the proliferation of human skin fibroblasts to a certain extent. This result shows that higher concentrations of grass carp egg polypeptide c can stimulate the proliferation process of human skin fibroblasts.

[0122] However, it is worth noting that although grass carp egg polypeptide C promoted the proliferation of skin fibroblasts at high concentrations, its effect was not as significant as its effect on collagen gene expression. This suggests that grass carp egg polypeptide C is relatively weak in stimulating fibroblast proliferation, and its main biological effect may not be concentrated on the enhancement of cell proliferation, but more on promoting the expression of collagen genes to exert its function.

[0123] Example 8

[0124] This embodiment of the present invention detects the effect of grass carp egg polypeptide C on the collagen level of aging mouse skin. Since hydroxyproline content is usually positively correlated with collagen content, hydroxyproline can be used as a reliable marker of collagen expression level. The present invention selects to detect the hydroxyproline content in mouse skin to characterize the collagen content.

[0125] After female KM mice (weighing about 20 g) were adapted for one week, the hair on the back of the mice was cut with surgical scissors, and then 10% sodium sulfide was used for depilation;

[0126] After observing for 3 days without any special reaction on the mouse skin, the mouse aging model was established. 20 mg of D-galactose was injected subcutaneously for 42 consecutive days, and the control group was injected with an equal amount of normal saline.

[0127] The experiment was divided into 4 groups, with 6 mice in each group. The control group and the model group were gavaged with 5 ml of purified water every day, and the low-concentration and high-concentration groups were gavaged with 5 ml of 25 μg / mL grass carp egg polypeptide c and 150 μg / mL grass carp egg polypeptide c, respectively.

[0128] After 42 days of experimental treatment, the mice were fasted for 12 h and then sacrificed. The skin of the depilated area on the back was quickly removed with surgical scissors, the subcutaneous fat layer was removed, and the skin was rinsed with saline for later use.

[0129] Weigh 0.5 g of skin tissue, add 4.5 mL of pre-cooled saline, and use a tissue homogenizer to prepare skin tissue homogenate;

[0130] The hydroxyproline ELISA detection kit was used according to the kit instructions to detect the hydroxyproline content in the skin tissue homogenate, thereby characterizing the collagen level in the skin.

[0131] The results obtained in this embodiment are shown in Table 2:

[0132] Table 2 Effect of grass carp egg polypeptide c on hydroxyproline content in skin of aging mice induced by D-galactose

[0133]

[0134]

[0135] It can be clearly seen from the experimental data in Table 2 that the hydroxyproline content in the model group was significantly reduced compared with the control group. This change indicates that the mouse skin aging model used in this study has been successfully constructed.

[0136] Compared with the model group, the hydroxyproline content of the low concentration group (25 μg / mL grass carp egg polypeptide c) increased, indicating that low concentrations of grass carp egg polypeptide c can promote the synthesis of collagen in mouse skin to a certain extent, thereby slowing down the process of skin aging. However, due to the low concentration, its effect is relatively limited.

[0137] In the high-concentration group (150 μg / mL grass carp egg polypeptide c), the hydroxyproline content increased significantly, indicating that higher concentrations of grass carp egg polypeptide c can significantly promote the synthesis of mouse skin collagen and significantly offset D-galactose-induced skin aging.

[0138] In summary, grass carp egg polypeptide C can enhance the synthesis of skin collagen, thereby effectively slowing down the skin aging phenomenon caused by D-galactose. Therefore, grass carp egg polypeptide C can be prepared into a biological preparation that provides skin collagen expression to effectively delay the aging of skin cells.

Claims

1. A biological preparation for increasing the expression of collagen in skin cells, characterized in that: The biological preparation is a liquid biological preparation, and the biological preparation consists of grass carp egg polypeptide and water.

2. The biological agent according to claim 1, characterized in that The grass carp egg polypeptide is prepared by the following preparation method: Clean the fresh grass carp roe and refrigerate at 4°C; Refrigerated grass carp roe were quickly frozen in liquid nitrogen and then ground into powder; Degreasing the powder with supercritical CO2, and leaving the powder to stand until the CO2 is completely released; Distilled water was added at a solid-liquid ratio of 1:20, the pH value was adjusted to 8, and ultrasonic treatment was performed at 180 W ultrasonic power for 60 min; The supernatant was collected by centrifugation, the pH of the supernatant was adjusted to 3, and after standing at room temperature, the protein precipitate was collected by centrifugation; The protein precipitate was dissolved in distilled water to prepare a protein solution, and the pH value of the protein solution was adjusted to 7.5; Add flavor protease at an enzyme dosage of 4000U / g protein precipitate and perform enzymolysis at 50°C for 4 hours; The enzymatic hydrolysate is heated to terminate the enzyme activity, and the supernatant is collected by centrifugation; The supernatant was treated by an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the retentate a and the filtrate a were collected; The filtrate was processed through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the retentate b and the filtrate b were collected; The filtrate b is placed in a freeze dryer for vacuum freeze drying to obtain grass carp egg polypeptide.

3. The biological agent according to claim 2, characterized in that In the biological preparation, the concentration of grass carp egg polypeptide is 25 μg / mL-300 μg / mL.

4. The biological agent according to claim 3, characterized in that The skin cells are human skin fibroblasts; The collagen is type I collagen and type III collagen.

5. An application of a grass carp egg polypeptide in the preparation of a biological preparation for increasing the expression of collagen in skin cells, characterized in that: The grass carp egg polypeptide is prepared according to the preparation method described in claim 2.

6. The use according to claim 5, characterized in that: The skin cells are human skin fibroblasts, and the collagen is type I collagen and type III collagen; In the biological preparation, the concentration of grass carp egg polypeptide is 25 μg / mL-300 μg / mL.

7. An application of grass carp egg polypeptide in the preparation of a biological preparation for slowing down skin aging, characterized in that: The grass carp egg polypeptide is prepared according to the preparation method described in claim 2.

8. The use according to claim 7, characterized in that: The biological agent for slowing down skin aging achieves the effect of slowing down skin aging by promoting the expression of type I collagen and type III collagen; The skin aging is skin aging caused by D-galactose; In the biological preparation, the concentration of the grass carp egg polypeptide is 25 μg / mL-300 μg / mL.

9. An application of a grass carp egg polypeptide in the preparation of a biological agent for improving skin cell growth, characterized in that: The grass carp egg polypeptide is prepared according to the preparation method described in claim 2.

10. The use according to claim 9, characterized in that: The skin cells are human skin fibroblasts; In the biological preparation, the concentration of the grass carp egg polypeptide is 150-300 μg / mL.

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