Cosmetic composition as well as preparation method and application thereof

Abstract

The invention discloses a cosmetic composition which is mainly prepared from micromolecular collagen peptide, beta-glucan, beta-cyclodextrin, a laggera alata extract, a chamomile extract, essence and antibacterial peptide according to the mass ratio of (0.9 to 1.5) to (0.3 to 1.5) to (0.45 to 0.55) to (0.09 to 0.12) to (0.09 to 0.12) to (0.09 to 0.13) to (0.09 to 0.15). The micro-molecular collagen peptide is used as a raw material and can permeate into a corium layer to stimulate collagen regeneration and improve skin elasticity, micro-molecules and beta-glucan form a'transdermal anchoring network 'and enhance skin repair capacity, and other raw materials have a synergistic effect to construct an'anti-inflammatory-bacteriostatic' barrier and reduce the risk of stimulation to sensitive skin. The invention further discloses application of the cosmetic composition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a cosmetic composition and a preparation method and application thereof. BACKGROUND

[0002] Skin aging is a complex physiological process, which is mainly manifested as wrinkles, laxity, dryness and decreased elasticity. Supplementing collagen is a means to cope with skin aging problems. Collagen peptide is a peptide segment in the protein released after hydrolysis of collagen. Small molecule collagen peptide can be directly absorbed by the human body without decomposition, and can directly penetrate the intestinal wall into the blood circulation. In the prior art, the methods for preparing collagen peptide from animal skin include acid method, alkali method and enzyme method. The enzyme method has the advantages of mild reaction conditions and short reaction time, and has been widely concerned and researched.

[0003] However, the active collagen peptide obtained by the existing collagen peptide extraction method has a large molecular weight and a low hydroxyproline content, and it is necessary to develop a small molecule collagen peptide with reduced molecular weight which can be applied to cosmetics. SUMMARY

[0004] According to a first aspect of the present application, a cosmetic composition is provided, mainly composed of small molecule collagen peptide, beta-glucan, beta-cyclodextrin, six-corner chrysanthemum extract, chamomile extract, essence and antibacterial peptide in a mass ratio of (0.9-1.5):(0.3-1.5):(0.45-0.55):(0.09-0.12):(0.09-0.12):(0.09-0.13):(0.09-0.15).

[0005] In some embodiments, the cosmetic composition is mainly composed of small molecule collagen peptide, beta-glucan, beta-cyclodextrin, six-corner chrysanthemum extract, chamomile extract, essence and antibacterial peptide in a mass ratio of 1:1:0.5:0.1:0.1:0.1.

[0006] In the cosmetic composition of the present application, the small molecule collagen peptide and the beta-glucan form a hydrated gel network at pH=5.0-5.5, and perform transdermal anchoring. The small molecule collagen peptide penetrates the dermis layer to stimulate collagen regeneration and improve skin elasticity. The beta-glucan activates the Langerhans cells in the epidermis layer and enhances the barrier repair capacity. The essence and the beta-cyclodextrin synergistically remove the odor. Specifically, the essence reduces the odor of the small molecule collagen peptide. The beta-cyclodextrin is first blended with the odor masking agent (essence) to form an inclusion compound. After being mixed with other components, the inclusion compound embeds the essence and the hydrophobic end of the collagen peptide, removes the odor (ammonia smell and fishy smell), and improves the stability of the composition. The six-corner chrysanthemum extract and the chamomile extract have anti-inflammatory and soothing effects (inhibit TNF-alpha and IL-6 inflammatory factors), repair redness and sensitive skin, and the antibacterial peptide targets and inhibits propionibacterium acnes, reduces the dependence on preservatives, and together builds an "anti-inflammatory and antibacterial" barrier to reduce the risk of stimulating sensitive skin.

[0007] The six-ribbed chrysanthemum extract is an active ingredient concentrate obtained by extracting and concentrating the whole grass of the six-ribbed chrysanthemum (Chrysanthemum morifolium Ramat. Laggera alata (D. Don) Sch.Bip. ex Oliv.) by a solvent (such as water, ethanol, etc.). The six-ribbed chrysanthemum extract has anti-inflammatory, antibacterial, antioxidant effects and anti-tumor activity.

[0008] The chamomile extract is an active ingredient concentrate obtained by solvent extraction from the flowers or whole grass of the chamomile (Matricaria recutita L.). It has excellent soothing, anti-inflammatory and antioxidant capacity.

[0009] In some embodiments, the essence is selected from at least one of plant protein and pelargonium graveolens oil.

[0010] In some embodiments, in the small molecule collagen peptide, the proportion of peptide segments with a molecular weight of less than 1000 Da is 80%-100%, and the hydroxyproline content is 3%-10%.

[0011] In some embodiments, in the small molecule collagen peptide, the proportion of peptide segments with a molecular weight of less than 1000 Da is 85%-100%, and the hydroxyproline content is 7%-10%.

[0012] The small molecule collagen peptide has the following effects: (1) inhibiting tyrosinase activity; (2) promoting cell proliferation; (3) up-regulating the expression of Col1a1 gene; (4) down-regulating the expression of Cemip1 gene; (5) anti-inflammatory; (6) inhibiting cell aging. Moreover, it has low cytotoxicity and good penetration effect.

[0013] In some embodiments, the small molecule collagen peptide is prepared by the following steps: (1) The fresh skin material is cut into pieces, pretreated with a sodium bicarbonate solution for 25-35 min, then rinsed with deionized water, and stored in a cold storage; (2) 10%-20% papain, 8%-12% bromelain, 15%-25% trypsin, 8%-12% pepsin, 10%-20% alkaline protease, 20%-28% neutral protease and 4%-6% flavor protease are mixed to obtain a composite enzyme preparation; (3) the skin obtained in step (1) is subjected to high-pressure steam sterilization at a pressure of 110-140 kPa and a temperature of 120-122°C for 20-40 min to obtain a skin with a degree of hydrolysis of 75%-85%, then distilled water is added to the skin to obtain a glue slurry, a complex enzyme preparation is added to the glue slurry, and the reaction is carried out at a pH of 6.8-7.2 and a temperature of 50-55°C for 5.5-6.5 h, and then the reaction system is subjected to enzyme inactivation at 85-95°C; (4) after the reaction system is cooled to room temperature, the system is purified and centrifuged, and the clear supernatant is obtained; wherein the skin is at least one of pig skin, alligator skin, deer skin, sheep skin, and cow skin.

[0014] In some embodiments, in step (1), the area of the fragments is 1-3 cm 2 .

[0015] In some embodiments, in step (1), the sodium bicarbonate solution is a 1%-3% (w / v) sodium bicarbonate solution at 60-70°C. Preferably, the sodium bicarbonate solution is a 2% (w / v) sodium bicarbonate solution at 65°C.

[0016] In some embodiments, in step (1), the temperature for refrigerated storage is -5~-3°C. Preferably, the temperature for refrigerated storage is -4°C.

[0017] In some embodiments, in step (2), the complex enzyme preparation consists of 15% papain, 10% bromelain, 20% trypsin, 10% pepsin, 15% alkaline protease, 25% neutral protease, and 5% flavor protease by mass percentage.

[0018] In some embodiments, in step (3), the skin obtained in step (1) is subjected to high-pressure steam sterilization at a pressure of 120 kPa and a temperature of 121°C for 30 min to obtain a skin with a degree of hydrolysis of 80%.

[0019] In some embodiments, in step (3), the reaction is carried out at a pH of 7.0 and a temperature of 52°C for 6 h, and then the reaction system is subjected to enzyme inactivation at 90°C.

[0020] In some embodiments, in step (3), the pH is adjusted to 6.8-7.2 with a 0.9-1.1 mol / mL hydrochloric acid solution. Preferably, the concentration of the hydrochloric acid solution is 1.0 mol / mL.

[0021] In some embodiments, in step (3), the ratio of the skin to distilled water is 1: (1.5-2.5). Preferably, the ratio is 1:2.

[0022] In some embodiments, in step (3), the amount of the complex enzyme preparation is 0.7%-1.2% of the mass of the glue pulp. Preferably, the amount of the complex enzyme preparation is 0.9% of the mass of the glue pulp.

[0023] In some embodiments, in step (4), the purification method is as follows: first, the system is coarsely filtered to remove lipids, then finely filtered with filter paper, and finally, 3% (w / w) activated carbon is added to the system, and the system is stirred at 65°C for 30 min for decolorization. Specifically, the system is coarsely filtered with 200-300 mesh cotton cloth to remove oil and fat.

[0024] In some embodiments, in step (4), the centrifugation method is as follows: the reaction system is centrifuged at a speed of 3000-5000 r / min for 8-12 min. Specifically, the reaction system is centrifuged at a speed of 4000 r / min for 10 min.

[0025] According to a second aspect of the present application, there is provided use of the cosmetic composition in the preparation of a cosmetic.

[0026] In some embodiments, the dosage form of the cosmetic can be selected from at least one of a facial mask essence, a cream, and a lotion.

[0027] According to a third aspect of the present application, there is provided a facial mask essence, prepared by the following steps: S1, mixing water, β-glucan, and the water phase ingredients at 70-75°C and a speed of 300-400 rpm to obtain a mixed system; S2, cooling the mixed system to 50-55°C, adding a surfactant to the mixed solution, and stirring for 8-12 minutes, then adding a solubilizer, β-cyclodextrin, and a fragrance, and continuing to stir for 8-12 minutes; S3, cooling the mixed system to 41-45°C, adding small molecule collagen peptides to the mixed system at a speed of 200-250 rpm, and dispersing by stirring for 15-20 minutes; S4, cooling the mixed system to 35-40°C, adding a fermentation product filtrate to the mixed system, stirring for 3-6 minutes, then adding a chamomile extract and a six-corner chrysanthemum extract, and dispersing by stirring for 8-12 minutes; S5, adding an antibacterial peptide and a preservative to the mixed system, and obtaining the facial mask essence.

[0028] In some embodiments, the water phase ingredients are selected from at least two of disodium EDTA, glycerol, butylene glycol, and erythritol.

[0029] In some embodiments, the surfactant is a polyether-modified siloxane, and the solubilizer is PEG-40 hydrogenated castor oil.

[0030] In some embodiments, the fermentation product filtrate consists of galactosomal yeast-like fermentation broth, bifidobacterium fermentation broth, and yeast fermentation broth.

[0031] In some embodiments, the preservative is selected from at least one of phenoxyethanol, 1,2-hexanediol, p-hydroxyacetophenone, octyl glycol, 1,2-pentanediol, and ethylhexylglycerin. The beneficial effects of this invention are as follows: (1) The composition of the present invention uses small molecule collagen peptides as raw materials, which can penetrate into the dermis to stimulate collagen regeneration and improve skin elasticity. The small molecules and β-glucan form a "transdermal anchoring network" to enhance the skin's repair ability.

[0032] (2) The small molecule collagen peptides of the present invention can inhibit tyrosinase activity, promote cell proliferation, upregulate the expression of Col1a1 gene, downregulate the expression of CemiP1 gene, and have anti-inflammatory and cell senescence-inhibiting effects. Moreover, they have low cytotoxicity and good penetration, and can be used to prepare cosmetics with anti-wrinkle, firming and anti-aging effects on the skin. Attached Figure Description

[0033] Figure 1 This is a chromatogram showing the molecular weight distribution of the small molecule active collagen peptides in Example 1 of the present invention; Figure 2 The inhibition rate of kojic acid on tyrosinase activity; Figure 3 The inhibition rate of small molecule collagen peptides on tyrosinase activity; Figure 4 The cell viability of Hacat cells in each group; Figure 5 The diagram shows the state of Hacat cells in each group; Figure 6 The cell viability of THP-1 cells in each group; Figure 7 The curve showing the relationship between DPPH free radical scavenging rate and reaction time in the VE group; Figure 8 The curve showing the relationship between the DPPH free radical scavenging rate of small molecule collagen peptides and reaction time; Figure 9 The cell survival rate of ESF cells in each group. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0035] The method for preparing small molecule active collagen peptides of the present invention is as follows: (1) Fresh pigskin was cut into 1 cm x 1 cm pieces, pretreated with 2% (w / v) sodium bicarbonate solution at 65°C for 30 min, rinsed with deionized water, and stored in a refrigerator at -4°C for later use.

[0036] (2) 15% papain, 10% bromelain, 20% trypsin, 10% pepsin, 15% alkaline protease, 25% neutral protease, and 5% flavor protease were mixed to obtain a complex enzyme preparation.

[0037] (3) The cowhide was sterilized and partially hydrolyzed (degree of hydrolysis 80%) by high-pressure steam sterilization at 120 kPa and 121°C for 30 min, then distilled water was added to the homogenate at a solid-liquid ratio of 1:2, and then the complex enzyme preparation (0.9% of the mass of the homogenate) was added to the homogenate, and a controllable enzymatic reaction was carried out at pH 7.0 (adjusted with 1 mol / L hydrochloric acid solution) and 52°C for 6 h, and then the enzyme activity was terminated by heat treatment at 90°C for 15 min.

[0038] (4) After the system cooled to room temperature, the upper oil was first filtered out with a 200-mesh cotton cloth, then filtered with filter paper, then 3% (w / w) activated carbon was added to the filtrate, and the mixture was stirred at 65°C for 30 min to decolorize, and finally centrifuged at 4000 r / min for 10 min, and the clear supernatant was obtained.

[0039] The molecular weight chromatogram of the obtained small molecule active collagen peptide is shown in Figure 1 The small molecule active collagen peptide has 83.69% of the peptide segment with a molecular weight less than 1000 Da, a hydroxyproline content of 9.32%, and a mass average molecular weight M w 927.

[0040] Example 1 This example provides a cosmetic composition, the formula of which is shown in Table 1.

[0041] Table 1 Formula components

[0042] Example 2 This example provides a cosmetic composition, which is different from Example 1 in that the amount of small molecule collagen peptide added in the formula is 0.9 parts by mass, the amount of β-glucan added is 0.8 parts, the amount of β-cyclodextrin added is 0.45 parts, the amount of six-ribbed chrysanthemum extract added is 0.09 parts, the amount of chamomile extract added is 0.09 parts, the amount of antimicrobial peptide added is 0.09 parts, and the amount of portulaca grandiflora oil added is 0.09 parts.

[0043] Example 3 The present example provides a cosmetic composition, which is different from Example 1 in that the amount of small molecule collagen peptide added in the formula is 1.5 parts by mass, the amount of β-glucan added is 1.5 parts, the amount of β-cyclodextrin added is 0.55 parts, the amount of six-edged chrysopsis extract added is 0.12 parts, the amount of chamomile extract added is 0.12 parts, the amount of antibacterial peptide added is 0.15 parts, and the amount of pelargonium capitatum oil added is 0.13 parts.

[0044] Example 4 The present example provides a preparation method of a mask essence, which comprises the following steps: (1) Water phase preparation: add water to a stainless steel reaction kettle, start stirring at a speed of 300-400 rpm, and heat to 70-75°C. Then add the β-glucan and the water phase ingredients (EDTA disodium, glycerol, butanediol, erythritol) one by one, stir for 5-10 minutes to completely dissolve after each addition, and then continue stirring at 70-75°C for 15 minutes to obtain a mixed solution; (2) Oil phase / solubilizing system addition: when the temperature of the reaction kettle drops to 50-55°C, add the surfactant bis-PEG-18 methyl ether dimethyl silane to the reaction kettle, then stir for 10 minutes, and then add the pre-dissolved mixture of pelargonium capitatum oil, β-cyclodextrin and PEG-40 hydrogenated castor oil to the reaction kettle, continue to stir for 10 minutes to ensure that the oil phase is completely dispersed (the system has no oil beads floating up); (3) High molecular active ingredient addition: when the temperature of the reaction kettle drops to 41-45°C, add the pre-treated sodium hyaluronate-glycerol dispersion liquid, slowly stir (reduce the speed to 200-250 rpm to avoid generating a large amount of bubbles), and stir for 15-20 minutes to completely dissolve. At this time, the system is transparent or translucent, and there is no flocculent material; then add soluble collagen and small molecule collagen peptide one by one, and stir for 5 minutes each time to ensure uniform dissolution; (4) Fermentation product, plant active ingredient addition: when the temperature of the reaction kettle drops to 35-40°C, add the galactosaccharomyces fermentate, schizosaccharomyces fermentate and yeast fermentate to the reaction kettle one by one, and then stir for 5 minutes; then add the chamomile extract and six-edged chrysopsis extract, and stir for 10 minutes to ensure uniform distribution of the active ingredients; (5) pH adjustment and preservative system addition: adjust the pH to 4.5-6.5 (consistent with the physiological pH of the skin, reduce irritation, and ensure the preservative effect) by adding hydroxyethylpiperazine ethane sulfonic acid solution (pre-prepared at a concentration of 5%); add the pre-prepared antibacterial peptide and preservative ingredients (add p-hydroxyphenylacetone, phenoxyethanol, octisalate, ethylhexylglycerin) one by one, and stir for 10 minutes to ensure uniform dispersion of the preservatives.

[0045] (6) Homogenization, filtration and degassing (enhancing skin feel and purity) Homogenization: Turn on the homogenization function of the reaction kettle (rotation speed is 1000-1500 rpm), homogenize for 5-8 minutes, break the possible small particles, and make the system more delicate; Filtration: Filter the system through a 0.45 μm microporous filter (use a stainless steel filter to pressurize filtration) to remove undissolved small impurities or precipitates; Degassing: Transfer the filtered serum into a degassing tank, turn on the vacuum degassing (vacuum degree is -0.08 to -0.09 MPa), and degas for 10 minutes to remove the residual bubbles in the system (to avoid the mask bag from swelling after filling, affecting the appearance).

[0046] In the mask serum of this embodiment, by mass percentage, small molecule collagen peptide 0.9%, β-glucan 0.8%, β-cyclodextrin 0.45%, six-angled sunflower extract 0.09%, chamomile extract 0.09%, antibacterial peptide 0.09%, disodium EDTA 0.03%, glycerol 8%, butanediol 4%, erythritol 3%, galactose yeast-like fungus fermentation broth 3%, Schizosaccharomyces fermenting broth 3%, Saccharomyces fermenting broth 3%, phenoxyethanol 0.5%, octisalate 0.2%, ethylhexylglycerin 0.5%, sodium hyaluronate 0.3%, soluble collagen 1%, PEG-40 hydrogenated castor oil 0.5%, bis-PEG-18 methyl ether dimethyl silane 1%, and the balance is water.

[0047] It should be noted that in this embodiment, the galactose yeast-like fungus fermentation broth is from Germany CLR, the active content (polysaccharide + polypeptide) is ≥3.5%; the Schizosaccharomyces fermentation broth is from Germany BASF, the active content (prebiotics + nucleotides) is ≥2.8%; and the Saccharomyces fermentation broth is from the Netherlands DSM, the active content (amino acid + organic acid) is ≥4.2%. The galactose yeast-like fungus fermentation broth uses Galactomyces fermentum (galactomyces) as the fermentation strain, and the strain has a unique galactose metabolism gene. The Saccharomyces fermentation broth uses Saccharomyces cerevisiae (brewer's yeast) as the fermentation strain.

[0048] Experimental Example 1 In this experimental example, the small molecule collagen peptide is subjected to B16 cell tyrosinase activity detection. The mouse B16 cells are stimulated by α-melanocyte stimulating hormone (α-MSH) to model the cells, and the sample to be detected is added for detection to evaluate whether the sample has an inhibitory effect on the cell tyrosinase activity.

[0049] (1) Cells: B16 cells (from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences).

[0050] (2) Reagents The reagents used in the experiment and their sources are shown in Table 2.

[0051] Table 2 Reagents and sources

[0052] (3) Cytotoxicity test The experiment was divided into blank group, positive group and drug group. The blank group was cultured with complete culture medium, the positive group was cultured with complete culture medium containing 10% DMSO, and the drug group was prepared with complete culture medium to prepare sample working solution with small molecule collagen peptide concentration of 50, 10, 2, 0.4, 0.08 mg / mL.

[0053] The B16 cell suspension with a confluence of 80%-90% was collected in a T25 bottle, counted, and the cell density was adjusted to inoculate a 96-well plate. The plate was cultured in a carbon dioxide incubator for 24 h. The culture medium in the wells was discarded, and the corresponding sample working solution was added according to the grouping. The 96-well plate was placed in the incubator for 24 h. Photographs were taken under a microscope. MTT working solution (1 mg / mL) was prepared. The culture solution was aspirated, 50 μL of MTT was added per well, and it was cultured in the dark for 2 h. The supernatant was discarded, and 100 μL of DMSO was added to dissolve the crystals. The shaking bed was shaken for 10 min. The absorbance value of each well at 570 nm wavelength was measured in the enzyme marker instrument. The cell survival rate of each group was calculated.

[0054] When the detection concentration of small molecule collagen peptide was 50, 10, 2, 0.4, 0.08 mg / mL, the cell survival rate was 71.71%, 90.13%, 100.28%, 96.87% and 101.64%, respectively. The CV90 was calculated to be 14.318 mg / mL.

[0055] (4) Experimental grouping The experimental grouping is shown in Table 3. The sample was diluted with DMEM to the sample concentration.

[0056] Table 3 Experimental grouping

[0057] (5) Experimental steps The cell suspension was prepared, the cell density was adjusted, and the cells were inoculated in 6-well plates and cultured in a carbon dioxide incubator for 24 h; then the culture plates were taken out, the culture solution in the holes was discarded, and different concentrations of the test substance and the culture medium of a-MSH were added to the corresponding holes, and the culture was continued in the carbon dioxide incubator for 72 h; then the culture plates were taken out, the culture solution was discarded, and each hole was washed twice with PBS; 1% Triton X-100 was added to the hole, the cells in each hole were collected with a cell scraper, and then the cells were frozen-thawed at -80°C, the cells were lysed, and the cell supernatant was obtained by centrifugation at 4°C; after the cell supernatant of each group was added to a 96-well plate, levodopa solution was immediately added, and then the mixture was mixed gently, and the reaction was carried out at 37°C in the dark for 2 h; and then the absorbance value was measured at 490 nm. The relative activity of tyrosinase was calculated according to the following formula.

[0058] Relative activity of tyrosinase (%) = (OD 490e / OD 490b ) x 100% In the formula, OD 490e is the absorbance of the test sample group and the positive control group; and OD 490b is the absorbance of the model control group.

[0059] (6) Experimental results The detection results of the relative activity of tyrosinase are shown in Table 4, and * indicates that there is a significant difference compared with the M group (P<0.05). As can be seen from Table 4, when the concentration of the small molecular collagen peptide is 10 mg / mL and 5 mg / mL, the relative activity of tyrosinase is 79.11% and 84.36%, respectively, which is significantly lower than that of the M group. However, at the detection concentrations of 1 mg / mL and 0.2 mg / mL, the relative activity of tyrosinase has no obvious difference with the M group, indicating that the sample has an inhibitory effect on the activity of B16 cell tyrosinase at the concentrations of 10 mg / mL and 5 mg / mL.

[0060] Table 4 Relative activity of tyrosinase of each group

[0061] Experimental Example 2 In this experimental example, the activity of tyrosinase was detected by a biochemical method.

[0062] (1) Reagents The reagents used in the experiment and their sources are shown in Table 5.

[0063] Table 5 Reagents and sources

[0064] (2) Experimental grouping The experimental grouping is shown in Table 6. The positive group and the sample group at each detection concentration are each provided with a corresponding background control group (using an equal amount of buffer instead of tyrosinase as a background control).

[0065] Table 6 Experimental grouping

[0066] (3) Sample preparation Sodium phosphate dibasic-citric acid buffer solution with pH = 6.8: 0.2 mol / L Na2HPO4·12H2O 154.5 mL was added to 0.1 mol / L citric acid monohydrate 45.5 mL to prepare 200 mL sodium phosphate dibasic-citric acid buffer solution, and the pH was adjusted to 6.8.

[0067] Positive control (kojic acid): diluted with buffer to a concentration of 1 mg / mL, 0.2 mg / mL, 0.04 mg / mL, and 0.008 mg / mL.

[0068] Levodopa solution: prepared with buffer to 1 mg / mL, and stored in the dark. Each sample: prepared with buffer to different concentrations of small molecule collagen peptides for standby.

[0069] (4) Experimental procedure Table 7 Well plate arrangement

[0070] A clean 96-well plate was taken, and the well plate arrangement was performed according to Table 7; the well plate was sequentially sampled, and after the sample and tyrosinase were sampled, 37°C incubation was performed for 10 min; levodopa solution was added to each well, and the reaction time of each well was controlled for 5 min; the temperature of the enzyme marker was set to 26°C, the detection wavelength was 475 nm, the reading was set to shake for 10 s before reading, and the OD475 value was detected after 5 min of reaction.

[0071] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1- (T-T0) / (C-C0)) x 100%, wherein T is the sample well absorbance value, i.e. the absorbance value of the solution after the sample and tyrosinase reaction; T0 is the sample background absorbance value; C is the tyrosinase background reaction well absorbance value, i.e. the solution absorbance value of tyrosinase and levodopa without sample; and C0 is the solvent background absorbance value.

[0072] (5) Results The tyrosinase activity inhibition rate is shown in Table 8, Figures 2-3 . Figure 2 The kojic acid tyrosinase activity inhibition rate is, Figure 3 The small molecule collagen peptide tyrosinase activity inhibition rate is, Figure 2 , Figure 3As shown in Table 8, the linear correlation coefficient between the inhibition rate of small molecule peptides on tyrosinase activity and the concentration curve is higher than 0.94. Based on the above data, the IC50 of the samples was calculated using linear fitting. 50 Value, predicting the IC50 value of small molecule collagen peptides 50 The concentration was 310.9 mg / mL.

[0073] Table 8 Tyrosinase activity inhibition rate

[0074] Experiment Example 4 This experimental example uses small molecule collagen peptides to detect their cellular metabolic activity (CCK-8).

[0075] (1) Cells: Human immortalized epidermal (Hacat) cells, purchased from China Center for Type Culture Collection.

[0076] (2) Reagents The reagents used in the experiment and their sources are shown in Table 9.

[0077] Table 9 Reagents and Sources

[0078] (3) Toxicity testing The study included a blank control (NC), a positive control (PC), and a drug-treated group. The blank control group was cultured in complete culture medium, the positive control group was cultured in complete culture medium containing 10% DMSO, and the drug-treated group had its small molecule collagen peptide diluted with 1×DMEM to the required detection concentration (V / V), with a total of 0.5 mL of sample working solution prepared. The concentrations of the small molecule collagen peptide were 125, 25, 5, 1, 0.2, and 0.04 mg / mL, respectively.

[0079] Cell viability of Hacat cells in each group as follows: Figure 5 As shown, the cell viability of small molecule collagen peptides at concentrations of 250, 50, 10, 2, 0.4, and 0.08 mg / mL was 14.87%, 85.64%, 91.08%, 87.09%, 71.53%, and 71.76%, respectively. The concentration of small molecule collagen peptides corresponding to a cell viability of 90% was 8.148 mg / mL.

[0080] (4) Experimental grouping The experimental groups are shown in Table 10. Samples were diluted to the required detection concentration (V / V) using DMEM to prepare a total of 0.7 mL of working solution.

[0081] Table 10 CCK-8 Experimental Grouping

[0082] (4) Experimental steps Hacat cells were digested; resuspended and counted, and the cell density was adjusted to 2.5 x 10 4 cells / mL, and the cells were added to a 96-well plate; the 96-well plate was placed in an incubator and cultured for 24 h. The culture medium in the wells was discarded, and the corresponding sample working solution was added according to the grouping; the 96-well plate was placed in an incubator and cultured for 72 h. Then, photographs were taken under a microscope; a CCK-8 working solution was prepared; the culture solution was aspirated, and 100 μL of the CCK-8 working solution was added per well, and the plate was cultured in the dark for 2 h; the absorbance value of each well at 450 nm was measured in an enzyme marker.

[0083] The cell proliferation rate of each group was calculated according to the following formula: cell proliferation rate = OD 450e / OD 450b , wherein OD 450e is the absorbance of the experimental sample group and the positive control group; and OD 450b is the absorbance of the blank control group.

[0084] (5) Experimental results The Hacat cell proliferation rate statistical results are shown in Table 11. As can be seen from Table 11, the cell proliferation rates of the Hacat cells under the action of the small molecule collagen peptide at concentrations of 25 mg / mL, 5 mg / mL, 1 mg / mL, and 0.2 mg / mL were 126.5%, 254.3%, 243.7%, and 141.5%, respectively, which were significantly higher than that of the blank control group (P < 0.05). The Hacat cell state diagrams of the various groups are shown in Figure 5 . As can be seen from Figure 5 , compared with the blank group, the cell number of the 25, 5, 1, and 0.2 mg / mL small molecule collagen peptide groups was significantly higher. The above results show that the small molecule collagen peptide has a promoting effect on the proliferation of Hacat cells.

[0085] Table 11 Hacat cell proliferation rate statistical results

[0086] Experimental Example 5 In this experimental example, the small molecule collagen peptide was subjected to ECM protein and hyaluronic acid metabolism.

[0087] (1) Reagents The reagents used in the experiment and their sources are shown in Table 12.

[0088] Table 12 Reagents and sources

[0089] (2) Experimental grouping The experimental grouping is shown in Table 13. The sample was diluted to the required detection concentration (V / V) using 1x DMEM, and a total of 2 mL of sample working solution was prepared.

[0090] Table 13: Experimental grouping of cell ECM protein and hyaluronic acid metabolism

[0091] (3) Experimental procedure The ESF cell suspension with a confluence of 80-90% was collected in a T75 bottle, counted, and adjusted to the cell density for inoculation in a culture plate, and cultured in a carbon dioxide incubator. The culture plate was removed, the culture medium in the wells was discarded, and different concentrations of working solution were added according to Table 13, and the culture was continued in the carbon dioxide incubator. The culture plate was removed, the culture medium in the wells was discarded, 1 mL of PBS was added to each well, and then discarded after rinsing, and the process was repeated once. The liquid in the wells was aspirated and placed on ice for standby, or treated with trypsin digestion, and the cells were collected and stored at -80°C. The total RNA was extracted according to the instructions of the Cell / Bacterial Total RNA Extraction Kit. The reverse transcription was performed according to the instructions of the First-Strand cDNA Synthesis Kit II, and the cDNA was obtained. The expression of related genes in cells was detected by fluorescence quantitative PCR. ® II First-Strand cDNA Synthesis Kit II to obtain cDNA. The expression of related genes in cells was detected by fluorescence quantitative PCR.

[0092] (4) Results The qPCR detection results are shown in Table 14. As can be seen from Table 14, the Col1a1 gene expression of the 5 mg / mL small molecule collagen peptide sample group was significantly up-regulated compared with the blank control group, about 125.4% of the blank control group, indicating that the 5 mg / mL small molecule collagen peptide sample could up-regulate the Col1a1 gene expression and could be used as evidence to support the anti-wrinkle efficacy claim of the sample. The Cemip1 expression of the 25 mg / mL small molecule collagen peptide group was significantly down-regulated compared with the blank control group, about 48.0% of the blank control group, indicating that the 25 mg / mL small molecule collagen peptide could down-regulate the Cemip1 gene expression and could be used as evidence to support the firming efficacy claim of the sample.

[0093] Table 14: qPCR detection results and relative expression amount calculation

[0094] Experimental Example 6 In this experimental example, the small molecule collagen peptide was subjected to THP-1 cell inflammatory factor expression detection.

[0095] (1) Cells: THP-1 cells (China Typical Culture Collection Center, number: GDC0100).

[0096] (2) Toxicity detection The study was divided into a blank group (NC), a positive group (PC), and a drug-treated group. The blank group was cultured in complete culture medium, the positive group was cultured in complete culture medium containing 10% DMSO, and the drug-treated group was prepared with small molecule collagen peptides in complete culture medium to prepare test solutions with small molecule collagen peptide concentrations of 125, 25, 5, 1, 0.2, and 0.04 mg / mL.

[0097] Each group of culture medium was seeded into a 96-well plate, 100 μL per well; 100 μL of a solution containing 8 × 10⁸ g of culture medium was added to each well of the 96-well plate. 4 Cell suspension of 100 cells; incubate in an incubator for 24 h; remove 96-well plates from the incubator for cell morphology observation and record photographs; add CCK-8, incubate in the dark for 50 min, shake on a shaker for 40 min; measure absorbance at 450 nm on an ELISA reader and calculate cell viability.

[0098] The survival rate of THP-1 cells in each group is as follows: Figure 6 As shown, the cell viability of THP-1 cells at concentrations of 125, 25, 5, 1, 0.2, and 0.04 mg / mL were 27.15%, 115.62%, 121.51%, 104.3%, 106.37%, and 93.92%, respectively. The small molecule collagen peptide concentration corresponding to a cell viability of 90% was 39.487 mg / mL.

[0099] (3) Experimental grouping First, an in vitro inflammatory cell model was established using lipopolysaccharide (LPS) as the model group (Group M). The groupings for the THP-1 cell inflammatory factor experiment are shown in Table 15. Appropriate amounts of samples were pre-diluted to four times the concentration to be tested as sample storage solutions. The sample storage solutions were then diluted twofold using 2X RPMI 1640 complete medium to serve as the working solutions for the anti-inflammatory and soothing efficacy tests.

[0100] Table 15 Experimental grouping of THP-1 cell inflammatory factors

[0101] (4) Experimental steps To simulate the inflammatory response of LCs in the skin to skin irritants, the prepared working solutions for each sample were seeded into 24-well plates. An equal volume of cell suspension was added to each well of the 24-well plates, and the plates were incubated for approximately 24 hours. LPS working solution was then added to the corresponding wells of each sample, and the plates were incubated for approximately 24 hours. The cell suspensions from each well were transferred to centrifuge tubes, centrifuged, and the supernatant was discarded. The cells were washed once with PBS to obtain cell pellet samples. Total RNA was extracted from each cell pellet sample using a cultured cell / bacterial total RNA extraction kit. HiScript was used to extract the total RNA. ®II The total RNA of each sample was reverse transcribed into cDNA by using the first strand cDNA synthesis kit; qPCR was used to detect and calculate the relative expression of TNF-a and IL-6 in each group of cDNA samples (2-ΔΔCq) with GAPDH as the internal reference; the relative expression (Mean±SD) of sample groups and model groups was statistically analyzed (t test) in Graphpad Prism 8.

[0102] (5) Results The relative expression of TNF-a and IL-6 (normalized to the M group) is shown in Table 16. Among them, "*" indicates that the relative expression is significantly lower than that of the model group, and the difference is statistically significant (p<0.05). As can be seen from Table 16, the relative expression of TNF-a and IL-6 of 25 mg / mL small molecule collagen peptide is about 65.3% and 7.6% of the model group, respectively, the relative expression of TNF-a and IL-6 of 5 mg / mL small molecule collagen peptide is about 91.8% and 32% of the model group, respectively, the relative expression of TNF-a and IL-6 of 1 mg / mL small molecule collagen peptide is about 87.7% and 106.4% of the model group, respectively, the relative expression of TNF-a and IL-6 of 0.2 mg / mL small molecule collagen peptide is about 99.8% and 105% of the model group, respectively, and the relative expression of TNF-a and IL-6 of 25 mg / mL small molecule collagen peptide is significantly lower than that of the model group (p<0.05), and the relative expression of IL-6 of 5 mg / mL small molecule collagen peptide is significantly lower than that of the model group (p<0.05). The above results show that small molecule collagen peptide has anti-inflammatory effect.

[0103] Table 16 Relative expression of TNF-a and IL-6 in each group

[0104] Experimental Example 7 In this experimental example, a parallel artificial membrane (PAMPA) permeation experiment was performed.

[0105] (1) Reagents The reagents used in the experiment and their sources are shown in Table 17.

[0106] Table 17 Reagents and sources for parallel artificial membrane permeation experiment

[0107] (2) Operation steps The small molecule collagen peptide was added to the donor well at a concentration of 500 mg / mL, and detection was performed at 0.5, 1, 2, 4, 6, 8 h. Specifically, before use, the pre-coated PAMPA plate system was taken out from -20℃ and placed at room temperature for at least 30 min. 300 μL of sample complex solution was added to each well in the donor plate. 200 μL of PBS buffer was added to each well in the receptor plate; then the pre-coated filter plate was placed on the receiving plate, and the pre-coated receptor plate was slowly lowered until it was placed on the receiving plate. The assembly was incubated at room temperature, and samples were taken at different incubation time points. According to the characteristics of the sample, the sample containing hydroxyproline was pre-determined, and the hydroxyproline concentration in the sample in the donor plate and the receptor plate was determined by the chloramine T method (using the hydroxyproline concentration instead of the sample concentration) respectively. The remaining protein sample without hydroxyproline was determined by the BCA method to determine the protein concentration of the receiving liquid and the feeding liquid at each time point respectively. The sample concentration in the two plates was determined, and the permeability of each test substance was calculated using the following formula: , wherein, C equilibrium = [C D (t) × V D + C A (t) × V A ] / (V D + V A ), the filter area A = 0.3 cm 2 , t is the incubation time (h); C A (t) is the hydroxyproline / protein concentration in the receptor well at t incubation time; the volume of the donor well V D = 0.3 mL; the volume of the receptor well V A = 0.2 mL.

[0108] (3) Results The detection results of the hydroxyproline content after the permeation of the small molecule collagen peptide are shown in Table 18. It can be seen that the hydroxyproline concentration in the receiving hydrolysate after the permeation of the small molecule collagen peptide (hydroxyproline content 10%) for 0.5 h to 8 h is 0.0617, 0.0278, 0.1019, 0.1188, 0.0247 and 4.0293 μg / mL respectively. As can be seen from the detection OD value in the detection standard curve, the OD value is between 0.0536 and 0.0588 when the hydroxyproline concentration is between 0 and 0.25 μg / mL. Although the OD value increases slightly with the increase of the hydroxyproline concentration, the increase is very small, and the OD value is very close to the blank. In this case, the detected hydroxyproline content is extremely low and fluctuates greatly. It can be confirmed that there is absolute hydroxyproline in the receiving liquid after 8 h of permeation, i.e. for the small molecule collagen peptide, the sample penetrates the parallel artificial semi-permeable membrane into the receiving well after 8 h of permeation, and the permeability of the sample is the highest at this time.

[0109] Table 18 Hydroxyproline content detection results after small molecule collagen peptide penetration

[0110] Experimental Example 8 In this experimental example, in vitro cell aging test was performed on small molecule collagen peptides.

[0111] (1) Cells: ESF-M (immortalized human embryonic skin fibroblasts), constructed by Guangdong Chikeng Medical Technology Co., Ltd.

[0112] (2) Reagents The reagents used in the experiment and their sources are shown in Table 19. An appropriate amount of each sample was pre-diluted to 2 times the concentration to be tested as a sample storage solution. The sample storage solution was diluted 2 times with 2x DMEM complete medium as the test sample working solution, which was prepared immediately before use.

[0113] Table 19 Reagents and sources for parallel artificial semi-permeable membrane penetration experiment

[0114] (3) Experimental grouping The in vitro cell aging test grouping is shown in Table 20.

[0115] Table 20 In vitro cell aging test grouping

[0116] (4) Experimental steps Take ESF-M cells in good growth condition and in the late logarithmic growth phase, digest the cells and count them. Adjust the cell density and inoculate the cells into a 24-well cell culture plate, then place it in a cell culture incubator for 18-24 h. Discard the original culture medium in the well, add the corresponding medium to each group according to the above test grouping, and continue to culture in the cell culture incubator for 24 h. Take the cell plate, aspirate the supernatant, wash with PBS, and replace the NC group with maintenance medium, and the remaining groups with H2O2-containing maintenance medium for continued culture for 24 h. Discard the original culture medium in the well, add complete culture medium to each group, and continue to culture in the cell culture incubator for 72 h. Remove the cell culture medium, wash with PBS, and then fix the cells. After aspirating the fixing solution, wash the cells with PBS, add β-galactosidase staining working solution, seal, and incubate overnight in an electrically heated constant temperature incubator. Remove the staining solution, wash the cells with PBS, and then perform nuclear staining. Collect the white light pictures under the microscope and the corresponding fluorescent pictures of the stained nucleus, calculate the number of positive cells in 3 or more fields of view for each group, and calculate the positive rate.

[0117] The cell aging positive rate is calculated according to the following formula: .

[0118] The cell senescence positive rate under each field of view of each group was obtained respectively, and the average of the senescence positive rate under each field of view of each group was finally obtained as the average cell senescence positive rate of the group.

[0119] (5) Results The in vitro cell senescence test results are shown in Table 21. As can be seen from the table, the cell senescence positive rates of the cells treated with 5 mg / mL, 1 mg / mL and 0.2 mg / mL small molecular collagen peptides were 29.298%, 36.801% and 30.239%, respectively, which were significantly different from the model group (p<0.05). Therefore, it is believed that 5 mg / mL, 1 mg / mL and 0.2 mg / mL small molecular collagen peptides have an inhibitory effect on H2O2-induced human immortalized skin fibroblast cell senescence. The cell senescence positive rate of the cells treated with 25 mg / mL small molecular collagen peptide was 39.675%, which had an inhibitory effect but was not significant compared with the model group.

[0120] Table 21 H2O2-induced ESF-M cell senescence data

[0121] Experimental Example 9 This experimental example carries out DPPH free radical scavenging detection on small molecular collagen peptides.

[0122] (1) Reagents: DPPH (brand: Macklin), anhydrous ethanol, purified water.

[0123] (2) Experimental grouping The DPPH free radical scavenging experiment grouping is shown in Table 22. Among them, the positive control group and the sample group to be tested are each detected at different concentrations, and the corresponding background control group (using an equal amount of sample solvent instead of DPPH as the background control) is set.

[0124] Table 22 DPPH free radical scavenging experiment grouping

[0125] Positive control group (VE): 95% ethanol is used to dilute and dissolve into different concentration gradients to verify the test system.

[0126] Each sample to be tested: the sample is diluted to multiple concentrations with purified water for standby.

[0127] (3) Experimental steps Take a clean 96-well plate and arrange the wells according to the grouping in Table 23; add samples to the wells according to the table above, with aqueous solution and 95% ethanol controls for DPPH background and solvent background, respectively; after adding samples to all groups, the total volume of solution in each well is 200 μL, and shake gently; set the temperature of the microplate reader to 26℃ and the detection wavelength to 517nm, and scan once every 5 minutes.

[0128] Table 23 Experimental Operation Grouping

[0129] The clearance rate is calculated using the following formula: Scavenging rate (%) = [1-(T-T0) / (C-C0)]×100%, where T is the absorbance of the sample well, i.e., the absorbance of the solution after the sample reacts with DPPH; T0 is the background absorbance of the sample; C is the absorbance of the DPPH well, i.e., the absorbance of the DPPH solution without the sample; and C0 is the background absorbance of the solvent.

[0130] (4) Results The relationship between DPPH free radical scavenging rate and reaction time in the VE group is shown in the curve. Figure 7 As shown in the figure, the relationship between the DPPH free radical scavenging rate of small molecule collagen peptides and reaction time is as follows: Figure 8 As shown. IC50 of DPPH radical scavenging rate for each sample after 5 min of reaction. 50 As shown in Table 24. Table 24 shows that the IC50 of small molecule collagen peptides... 50 It was 96.17 mg / mL.

[0131] Table 24 IC50 of DPPH radical scavenging rate for each group 50

[0132] Experimental Example 10 This experimental example uses small molecule collagen peptides to detect ESF cytotoxicity.

[0133] (1) Cells: Human embryonic skin fibroblasts (ESF) cells: purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0134] (2) Experimental grouping The experimental groups for ESF cytotoxicity assay are shown in Table 25. The blank control group was cultured in complete culture medium, the positive control group was cultured in complete culture medium containing 10% DMSO, and the drug-treated groups were prepared with small molecule collagen peptides (samples) in complete culture medium to different concentrations of small molecule collagen peptides.

[0135] Table 25 Experimental Grouping for ESF Cytotoxicity Assay

[0136] (3) Experimental steps T25 bottle collection of ESF cell suspension of 80%~90% confluence, count, adjust the cell density to inoculate in 96-well plate culture plate, in the carbon dioxide incubator for 24 h; discard the culture medium in the hole, according to the group to add the corresponding culture medium; 96 well plate into the incubator for 24 h. Take a picture under a microscope; preparation of MTT working solution (1 mg / mL); suck the culture solution, add 50 μL MTT / well, avoid light for 2 h, suck the supernatant, add 100 μL DMSO to dissolve the crystal, shake bed shock 10 min; in the microplate reader to measure the absorbance value of each hole at 570 nm wavelength, calculate the cell survival rate.

[0137] (4) Results The cell survival rate of each group of ESF cells is shown in Table 26. As can be seen from Table 26, for small molecule collagen peptides, the cell survival rate is 73.81%, 87.91%, 97.38%, 103.88%, 106.00% and 110.95% from high to low, and the CV90 is calculated to be 9.820 mg / mL. The results are consistent with the observed phenomena under a microscope. Figure 9

[0138] Table 26 Cell survival rate

[0139] The above only describes some embodiments of the present application. For those skilled in the art, without departing from the inventive concept, several modifications and improvements can be made, which are all within the scope of protection of the present application.​

Claims

1. A cosmetic composition characterized in that, The small molecule collagen peptide, the beta-glucan, the beta-cyclodextrin, the six-edged chrysopsis extract, the chamomile extract, the essence and the antimicrobial peptide are mainly composed of mass ratio (0.9-1.5):(0.3-1.5):(0.45-0.55):(0.09-0.12):(0.09-0.12):(0.09-0.13):(0.09-0.15).

2. The cosmetic composition according to claim 1, characterized by In the small molecule collagen peptide, the peptide segment with a molecular weight less than 1000 Da accounts for 80-100%, and the hydroxyproline content is 3-10%.

3. The cosmetic composition according to claim 1 or 2, characterized by, The small molecule collagen peptide is prepared by the following steps: (1) After cutting fresh skin into pieces, the skin is pretreated with a sodium bicarbonate solution for 25-35 min, then the skin is rinsed with deionized water and stored in a cold storage; (2) 10-20% papain, 8-12% bromelain, 15-25% trypsin, 8-12% pepsin, 10-20% alkaline protease, 20-28% neutral protease and 4-6% flavor protease are mixed to obtain a composite enzyme preparation; (3) The skin obtained in step (1) is subjected to high-pressure steam sterilization at a pressure of 110-140 kPa and a temperature of 120-122℃ for 20-40 min to obtain skin with a degree of hydrolysis of 75-85%, then distilled water is added to the skin to obtain a glue slurry, and the composite enzyme preparation is added to the glue slurry, and the reaction is carried out at a pH of 6.8-7.2 and a temperature of 50-55℃ for 5.5-6.5 h, then the enzyme inactivation of the reaction system is carried out at 85-95℃; (4) After the reaction system is cooled to room temperature, the system is purified and centrifuged, and the clear supernatant is obtained. The skin is at least one of pig skin, alligator skin, deer skin, sheep skin and cow skin.

4. The cosmetic composition according to claim 3, characterized by In step (1), the sodium bicarbonate solution is a 1-3% (w / v) sodium bicarbonate solution at 60-70℃.

5. The cosmetic composition according to claim 3, characterized by In step (3), the ratio of skin to distilled water is 1:(1.5-2.5), and the amount of composite enzyme preparation is 0.7-1.2% of the mass of glue slurry.

6. The cosmetic composition according to claim 1, characterized by The essence is selected from at least one of plant protein and pelargonium capitatum oil.

7. Use of the cosmetic composition according to any one of claims 1 to 6 for the manufacture of a cosmetic product, characterized in that, The dosage form of the cosmetic is at least one of a mask essence, a cream and a lotion.

8. A mask serum, characterized by, The mask essence comprising the cosmetic composition of any one of claims 1-6 is prepared by the following steps: S1, mixing water, beta-glucan and water phase ingredients at 70-75℃ and a rotation speed of 300-400 rpm to obtain a mixed system; S2, cooling the mixed system to 50-55℃, adding a surfactant to the mixed solution, stirring for 8-12 minutes, and then adding a solubilizing agent, beta-cyclodextrin and essence, and continuing to stir for 8-12 minutes; S3, cooling the mixed system to 41-45℃, adding small molecule collagen peptide to the mixed system at a rotation speed of 200-250 rpm, and dispersing by stirring for 15-20 minutes; S4, cooling the mixed system to 35-40℃, adding the fermentation product filtrate to the mixed system, stirring for 3-6 minutes, then adding the chamomile extract and the six-angle chrysanthemum extract, and dispersing by stirring for 8-12 minutes; S5, adding the antibacterial peptide and the preservative to the mixed system, and obtaining the product.

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