Lily peptide and preparation method thereof

Abstract

The invention belongs to the field of cosmetics, particularly relates to a lily peptide and a preparation method thereof, and more particularly relates to a lily fermentation method and the lily peptide prepared by the lily fermentation method. The invention provides a preparation method of lily peptide. Compared with the greenish lily peptide obtained in the prior art, the greenish lily peptide prepared by the method disclosed by the invention has a stronger skin care effect, including a fibroblast proliferation effect and a cell scratch repairing effect.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics, and in particular, relates to a lily flower peptide and a preparation method thereof, and more particularly, relates to a lily flower fermentation method and the lily flower peptide prepared therefrom. Background Art

[0002] Circadian rhythms, 24-hour oscillations of behavior and physiology, are reflected in all cells to optimize cellular function and respond to day-night-related environmental challenges. This multi-oscillatory network is composed of the suprachiasmatic nucleus (SCN) located in the hypothalamus, which directs the rhythms of the organism to express physiological functions and behaviors through a hierarchical system. This system has been highly conserved throughout evolution and uses a transcription-translation autoregulatory cycle. This master clock follows environmental cues and regulates the organism's sleep patterns, body temperature, heart activity and blood pressure, hormone secretion, oxygen consumption, and metabolic rate. Mammalian peripheral clocks and clock gene expression have recently been discovered and are present in all nucleated cells of our body. Like other important organs of the body, the skin also has a cycle informed by this master regulator. In addition, skin cells have peripheral clocks that can work autonomously.

[0003] Modern lifestyles, including daytime sun avoidance / sun protection, cross-time zone flights, nighttime blue light pollution, staying up late or working night shifts, and even insomnia, cause the body and skin biological clock genes to lose synchronization, affecting the normal 24-hour circadian rhythm. Lifestyle changes-skin problems caused by biological clock disorders include damaged keratin barrier, dry and sensitive skin; weakened skin repair ability, premature skin aging; regulating clock genes, including Per, Cry, Clock, and Bmal-1, helps restore the circadian rhythm of skin cells, relieve dry, sensitive, and aging skin, and achieve multiple skin care effects such as soothing, repairing, and anti-aging, thereby helping contemporary people to more deeply and comprehensively deal with the adverse skin effects brought about by modern lifestyles.

[0004] Therefore, there is a need in the art for a product that can regulate clock genes and has multiple skin care effects such as soothing, repairing, moisturizing, repairing, anti-wrinkle, firming, and light protection, while also having high safety. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a method for preparing lily flower peptide, comprising the following steps:

[0006] S1. Crush, dissolve and filter the lily raw material to obtain a filtrate;

[0007] S2, adjusting the pH value of the filtrate until the precipitate is completely precipitated and filtering to obtain the precipitate;

[0008] S3, adding a composite enzyme to the precipitate for enzymolysis to obtain an enzymolysis product, wherein the composite enzyme comprises flavor protease and papain;

[0009] S4, fermenting the enzymatic hydrolysis product using bifidobacteria; and

[0010] S5. Obtain lily flower peptide.

[0011] The lily flower peptide prepared by the method of the present invention has a stronger effect on the proliferation of fibroblasts and the repair effect on cell scratches than the lily flower peptide obtained in the prior art.

[0012] Furthermore, in the step S1, water with a pH value of 8 to 12, preferably 10, is used for dissolution.

[0013] Furthermore, in the step S1, there is also a step of using ultrasonic stirring. In some specific embodiments, the ultrasonic is 300W ultrasonic.

[0014] Furthermore, in the step S2, the pH value of the filtrate is further adjusted to 3-5, preferably to 4.

[0015] Furthermore, in step S3, the concentration ratio of flavor protease:papain is (1-5):(5-1). Preferably, the concentration ratio of flavor protease:papain is 1:1.

[0016] Furthermore, in step S3, the concentration of the complex enzyme is 5000-20000 U / g, preferably 8000-15000 U / g, and more preferably 12000 U / g.

[0017] The term "complex enzyme concentration" refers to the ratio of the added complex enzyme to the precipitate for enzymatic hydrolysis. For example, 5000-20000 U / g means that 5000-20000 U of complex enzyme is added to 1 g of precipitate.

[0018] Further, in step S3, the conditions for the enzymolysis are a temperature of 50-60°C, a pH of 7-9, and a reaction time of 3-10 hours. In a specific embodiment, in step S3, the conditions for the enzymolysis are a temperature of 55°C, a pH of 8, and a reaction time of 5 hours.

[0019] Furthermore, in step S4, the bifidobacterium may be one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum, or Bifidobacterium thermophilum.

[0020] Furthermore, in step S4, the fermentation condition is 35-50° C., and the reaction time is 48-96 hours. In a specific embodiment, the fermentation condition is 45° C., and the reaction time is 72 hours.

[0021] Furthermore, in the step S5, it further includes inactivating the enzyme at 95° C. for 20 minutes; high-speed centrifugation to obtain the supernatant; passing the filtrate through an ultrafiltration membrane; and spray drying to obtain the lily flower peptide.

[0022] Furthermore, the peptide content of the obtained lily flower peptide is greater than 80%.

[0023] Furthermore, the lily flower peptides obtained include a polypeptide mixture as described in Table 1.

[0024] Specifically, the polypeptide mixture includes the following:

[0025] DQLSDLQR,VEIVDDIHAAIDHIHQ,ADTGGFLEV,MIIAVPTG,NFEGSIP,LLQDIVPTF,CIQGLPEG,AVEEAGGPK,PEDIAPNK,NLTDDDVV,S DEGDVLY, QKGDQYLNS, QAAASKAATMD, SAEAIVLIGTA, GIAVLLK, INEEAGDEIKEAS, SNVGGSGAVQSV, EKGGVFFC, DPSSFPP, WSPSMIK.

[0026] In a second aspect, the present invention provides a lily flower peptide, which is obtained by the preparation method as described above.

[0027] Furthermore, the peptide content of the lily flower peptide is greater than 80%.

[0028] Furthermore, the lily flower peptides obtained include a polypeptide mixture as described in Table 1.

[0029] Specifically, the polypeptide mixture includes the following:

[0030] DQLSDLQR,VEIVDDIHAAIDHIHQ,ADTGGFLEV,MIIAVPTG,NFEGSIP,LLQDIVPTF,CIQGLPEG,AVEEAGGPK,PEDIAPNK,NLTDDDVV,S DEGDVLY, QKGDQYLNS, QAAASKAATMD, SAEAIVLIGTA, GIAVLLK, INEEAGDEIKEAS, SNVGGSGAVQSV, EKGGVFFC, DPSSFPP, WSPSMIK.

[0031] In a third aspect, the present invention provides the use of the above-mentioned lily flower peptide for preparing skin care products.

[0032] Furthermore, the skin care product has multiple functions, including one or more of moisturizing, soothing, repairing, anti-wrinkle, firming, repairing, photoprotection or anti-photodamage.

[0033] In a fourth aspect, the present invention provides a skin care product with multiple functions, comprising the lily flower peptide as described above.

[0034] Furthermore, the multiple functions include one or more of moisturizing, soothing, repairing, anti-wrinkle, firming, repairing, photoprotection or anti-photodamage.

[0035] Furthermore, the products include lipstick, lip gloss, lip balm, lip mask, loose powder, honey powder, pressed cake, primer, foundation, air cushion, blush, contouring, eye cream, gel, eye shadow, makeup remover, cleanser, face cream, essence, lotion, emulsion, face cream, CC cream (ColorControl Cream).

[0036] Furthermore, the mass volume concentration of the lily peptide in lipstick, lip glaze, loose powder, pressed powder, primer, foundation, blush, contouring, eye shadow, makeup remover, and cleanser is 0.001-1%, that is, 0.001-1g of lily peptide is added to every 100mL.

[0037] Furthermore, the mass volume concentration of the lily flower peptide in essence, lotion, emulsion and cream is 0.01-0.5%, that is, 0.01-0.5g of lily flower peptide is added to every 100mL.

[0038] Furthermore, the product also includes one or more of an emulsifier, a surfactant, a carrier, a flavor, a preservative, and an antioxidant. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the sample scratch healing diagram;

[0040] Figures 2 to 4 The clock gene regulation effect of lily peptide;

[0041] Figure 5 The negative control was 0.9% NaCl, and the positive controls were 0.2%, 0.3%, and 0.5% sodium hydroxide solutions for CAM results;

[0042] Figure 6 This is the CAM result of the lily flower peptide of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0044] Example 1. Preparation of lily flower peptide

[0045] The raw material was crushed and added to water with pH 10; ultrasonic stirring at 300W for 3h; filtrate was obtained, and the pH value of the filtrate was adjusted to 4; precipitate was obtained by filtration, and the pH value was adjusted to 7; compound enzyme 12000U / g (flavor protease: papain = 1:1) was added; temperature 55℃, pH 8, reaction time 5h; bifidobacterium fermentation, 45℃, reaction time 72h; enzyme inactivation at 95℃ for 20min; high-speed centrifugation, supernatant was taken; filtrate was passed through ultrafiltration membrane; spray drying was performed to obtain lily peptide powder. The lily peptide was tested for its components, and its peptide content was >80%. It is a mixture of multiple short peptides, some of which are shown in Table 1. Its quality inspection is shown in Table 2.

[0046] Table 1

[0047]

[0048] Table 2

[0049]

[0050] According to the above preparation method, but the flavor protease and papain (complex enzyme) are replaced by alkaline protease (Alcalase alkaline protease, enzyme activity> 600,000 U / g, enzyme substrate ratio 5000 U / g; temperature 55°C, pH value to 8-8.5, reaction time 3h), it is also a mixture of multiple short peptides, some of which are shown in Table 3.

[0051] Table 3

[0052]

[0053]

[0054] LC-MS / MS analysis showed that the compound enzyme hydrolysis-lily peptide contained 535 peptides, with amino acid sequence lengths of pentapeptide-tetradeceptide, and molecular weight distribution <1000Da, accounting for 76.09.4%, >1000Da, accounting for 23.91%. Alkaline protease-lily peptide contained 1393 peptides, with amino acid sequence lengths of pentapeptide-tetradeceptide, and molecular weight distribution <2000Da, accounting for 35.4%; >2000Da, accounting for 64.6%. The comparison of peptides prepared by the two preparation methods is shown in Table 4. It can be seen from the table that the peptide mixtures prepared by the two methods are completely inconsistent.

[0055] Table 4

[0056]

[0057] Example 2: Comparison of the effects of lily peptides prepared by two methods

[0058] The cell efficacy test also showed that the lily flower peptide prepared by alkaline protease hydrolysis-bifidobacterium fermentation process can significantly promote the proliferation of HFF-1 fibroblasts in the concentration range of 0.125-1 mg / ml, improve cell viability, and has a repair effect; and the lily flower peptide prepared by composite enzymatic hydrolysis-bifidobacterium fermentation process can also significantly promote the proliferation of HFF-1 fibroblasts in the concentration range of 0.125-1 mg / ml, and its effect is better than the polypeptide mixture obtained by alkaline protease hydrolysis. The results are shown in Table 5.

[0059] Similarly, in the HaCaT human immortalized keratinocyte scratch model, compared with the blank control group, the lily flower peptide prepared by alkaline protease hydrolysis-bifidobacterium fermentation process can significantly promote cell scratch healing in the concentration range of 0.1-1 mg / ml; and the lily flower peptide prepared by composite enzymatic hydrolysis-bifidobacterium fermentation process can also significantly promote cell scratch healing in the concentration range of 0.1-1 mg / ml, and its overall effect is better than that of the lily flower peptide obtained by alkaline protease hydrolysis. The results are shown in Table 6, which also indicates that the composite enzyme-hydrolyzed lily flower peptide has a stronger skin care effect than the alkaline protease hydrolyzed lily flower peptide.

[0060] Table 5. Effects of two samples prepared by different enzymatic hydrolysis processes on the proliferation of HFF-1 human fibroblasts

[0061]

[0062] Table 6. The scratch repair effects of two samples with different enzymatic hydrolysis processes on HaCaT human immortalized keratinocytes

[0063]

[0064] Example 3. Antioxidant activity of lily peptides fermented by different strains

[0065] The fermentation was carried out according to the method described in Example 1, except that it was divided into three groups, including no fermentation, bifidobacterium-lactic acid bacteria fermentation and bifidobacterium fermentation, and samples were obtained respectively for DPPH antioxidant activity test. The results showed that the lily peptide obtained by bifidobacterium fermentation had an IC 50 The concentration of α-glutamic acid was the lowest and the antioxidant activity was the strongest. The results are shown in Table 7.

[0066] Table 7. DPPH radical scavenging rate of three process samples

[0067]

[0068]

[0069] Example 4: Scratch repairing effect of lily peptides fermented by different strains

[0070] The fermentation was carried out according to the method described in Example 1, except that it was divided into three groups, including no fermentation, bifidobacterium-lactic acid bacteria fermentation and bifidobacterium fermentation, and samples were obtained respectively, and the scratch repair efficacy of HaCaT human immortalized keratinocytes was tested. The results showed that the lily peptide obtained by bifidobacterium fermentation had the strongest scratch repair efficacy. The results are shown in Table 8.

[0071] Table 8. The scratch repair effects of three process samples on HaCaT human immortalized keratinocytes

[0072]

[0073] Example 5: Effect of different extraction methods on the effect

[0074] In the HFF-1 human fibroblast proliferation model, compared with the blank control group, lily peptide in the concentration range of 0.125-1 mg / ml can significantly promote cell proliferation and improve cell viability (P<0.05-0.01), and has a repair effect; and 0.125-1 mg / ml lily water extract or lily alcohol extract (70% ethanol) can also improve HFF-1 cell viability to a certain extent, but both are weaker than lily peptide, and compared with the blank control group, there is no significant difference (P>0.05). The results are shown in Table 9. Similarly, in the HaCaT human immortalized keratinocyte scratch model, compared with the blank control group, lily flower peptides in the concentration range of 0.1-1 mg / ml can significantly promote cell scratch healing (P<0.01); lily flower water extract has no significant effect on scratch healing. Although lily flower alcohol extract (70% ethanol) can significantly improve the scratch healing rate at a concentration of 1 mg / ml, its overall effect is weaker than that of lily flower peptides. The results are shown in Table 10, which all indicate that the preparation process of lily flower peptides is superior to that of traditional water-alcohol extraction of chemical components (polysaccharides, flavonoids, saponins, etc.).

[0075] Table 9 Effects of three samples with different preparation processes on the proliferation of HFF-1 human fibroblasts

[0076]

[0077]

[0078] Note: Lily flower water extract: 5g lily flower + 100g purified water, heat extraction at 60℃ for 1h, filter through 0.22μm filter membrane. Lily flower alcohol extract: 5g lily flower + 100g 70% ethanol, heat extraction at 60℃ for 1h, filter through 0.22μm filter membrane.

[0079] Table 10. Scratch repair effects of three process samples on HaCaT human immortalized keratinocytes

[0080]

[0081] Note: Lily flower water extract: 5g lily flower + 100g purified water, heat extraction at 60℃ for 1h, filter through 0.22μm filter membrane. Lily flower alcohol extract: 5g lily flower + 100g 70% ethanol, heat extraction at 60℃ for 1h, filter through 0.22μm filter membrane.

[0082] Example 6: Scratch protection test of the lily flower peptide of the present invention

[0083] Materials and methods

[0084] 1. Experimental cells: HACAT human immortalized keratinocytes.

[0085] 2. Main reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), TGF-β (MCE), ROS kit (Biyuntian).

[0086] 3. Main equipment: CO2 incubator (healforce), biological safety cabinet (healforce), fluorescence inverted microscope (OLYMPUS).

[0087] 4. Experimental methods

[0088] The sample cytotoxicity experiment and efficacy evaluation test were carried out according to the "QYGEMG-SOP-2005 HaCaT Immortalized Keratinocyte Scratch Test Standard Operating Procedure" of the Basic Research Department.

[0089] 4.1 Cytotoxicity assay

[0090] 1) Cell plating: dilute the cell suspension to a density of 2×10 5 Each well was inoculated into a 96-well plate at 100 μL / well and placed in a 37°C, 5% CO2 incubator for 24 h.

[0091] 2) Sample addition: Take out the 96-well plate, discard the supernatant, add DMEM complete medium to the normal control group, add DMEM complete medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 h;

[0092] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to balance, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value; calculate cell viability: cell viability (%) = sample OD value / blank OD value*100%

[0093] Note: Subsequent efficacy evaluation tests were performed when the cell viability was above 90%.

[0094] Efficacy evaluation

[0095] 1) Cell plating: dilute the cell suspension to a density of 6×10 5 Each well was inoculated into a 6-well plate, 2 mL / well, and then placed in a 37°C, 5% CO2 incubator for 24 h.

[0096] 2) Scratching and adding samples: Take out the 6-well plate, discard the supernatant, draw horizontal lines on the back of each well with a marker pen at the bottom of the well plate, draw 5 horizontal lines on average, add 1mL PBS to each well, draw a vertical straight line on the bottom of the 6-well plate with a gun tip, and slowly wash with PBS 3 times to remove the detached cells. Serum-free DMEM high-glucose medium was added to the control group, and DMEM high-glucose medium containing 2.5% FBS was added to the positive group, 2mL / well, and the test sample group was prepared with serum-free medium to the corresponding concentration. After the addition of drugs, it was placed in a 37°C, 5% CO2 incubator for 24h±2h.

[0097] 3) Photography: At 0 and 24 hours after administration, the scratches in the 6-well plate were placed under a microscope and photographed. The scratches were divided into 6 areas by the horizontal lines on the back. The visual field under each area was photographed. The positions of the scratches in the visual fields selected at different time points should be consistent. Each group had 6 different visual fields at each time point, i.e. 6 pictures.

[0098] 4) The area A of the image is calculated using the software that comes with the microscope.

[0099] The scratch healing rate is calculated as follows:

[0100]

[0101] The mean value (Mean) and standard deviation (SD) of each group of experiments were calculated, and Graphpad Prism software was used for data statistical analysis, and one-way Anova analysis was used for data between multiple groups. P < 0.05 was a significant difference, P < 0.01 was an extremely significant difference, and P > 0.05 was not a significant difference.

[0102] Test results: CCK8 test results of cytotoxicity of each sample: see Table 11. According to the CCK8 results, the lily flower peptide showed no cytotoxicity in the concentration range of 7.8 μg / ml-1 mg / ml.

[0103] Scratch healing rate results: see Figure 1 Compared with the blank group (KB), the scratch healing rate of the positive group (FBS2.5%) was significantly different from that of the blank group (KB) as shown in Table 12. Lily peptide, at 31-125 μg / ml, can significantly promote the healing of scratches, with healing rates of 80.91%, 83.81% and 87.58% (P<0.001), respectively, and has a repair effect; at a concentration of 31 μg / ml, lily peptide is better than carnosine and blue copper peptide.

[0104] Table 11. Cytotoxicity CCK8 test results of each sample

[0105]

[0106] Table 12. Sample scratch healing rate

[0107]

[0108] Note: Comparison between sample group and blank group (KB), ***p<0.001; **p<0.01; *p<0.05

[0109] Example 7: Test on the soothing effect of the lily flower peptide of the present invention

[0110] 1. Zebrafish test

[0111] Zebrafish 2 days after fertilization (2dpf) were divided into three groups, namely normal control group, model control group and test group. The normal control group and model group were not treated, and the test cosmetics were added to the test group (the test cosmetics were dissolved in the water for fish farming, and the water-soluble drug was administered). After incubation in the dark for 24 hours, the model control group and the test group were water-soluble and glacial acetic acid was administered to establish the zebrafish pain model. After incubation for a period of time, the movement distance of the zebrafish was detected using a zebrafish behavior analyzer to quantitatively evaluate the neurosoothing effect of the test product.

[0112] Test results:

[0113] As shown in Table 13, compared with the normal control group, the movement distance of the zebrafish model stimulated by glacial acetic acid increased significantly (P<0.001); compared with the model group, 0.1% lily peptide significantly reduced the movement distance of the zebrafish model (P<0.001), with an inhibition rate of 62%. Compared with the model control group, the total movement distance of zebrafish in the 0.1% lily peptide group was significantly reduced, indicating that the test product has a neurosoothing effect.

[0114] Table 13. Effect of 0.1% lily peptide on the movement distance of zebrafish stimulated by glacial acetic acid

[0115]

[0116] 2. Human keratinocyte (HaCaT) TRPV1 gene expression level test

[0117] Cells were seeded in 6-well plates (3×10 5 cells / well / 2mL), and incubate in a 37°C, 5% CO2 incubator overnight. Dosing: Remove the culture medium, add 2mL of culture medium to each well of the blank control group (BC), add cell culture medium containing the sample to be tested at the corresponding concentration to the sample group, and continue to culture in a 37°C, 5% CO2 incubator for 24 hours.

[0118] Detection:

[0119] A. After 24 hours of culture, remove the supernatant, wash the cell surface twice with PBS, add 1 mL Trizol, 200 μL chloroform, isopropanol, 75% ethanol, etc. in sequence, extract RNA according to the RNA extraction procedure, finally add 10 μL RNase-free water to dissolve RNA, and measure the total RNA concentration using a multifunctional microplate reader.

[0120] B. Reverse transcribe the above RNA into cDNA, and then The Premix EX TaqTM kit was operated according to the instructions. The target gene and the internal reference gene were amplified using a qRT-PCR instrument, the expression level of the TRPV1 gene in HaCaT cells was detected, the expression of the TRPV1 gene in HaCaT cells by the samples was calculated, and the soothing ability of the samples to be tested was evaluated.

[0121] Table 14. Effects of samples on TRPV1 gene expression levels in HaCaT cells

[0122]

[0123] Note: Comparison between model group and blank control group, ###p<0.001; comparison between sample group and model group, *p<0.05, ***p<0.001

[0124] Test results:

[0125] As shown in Table 14, compared with the blank control, the model group significantly promoted the relative expression of TRPV1 gene (p < 0.001), and the relative expression of TRPV1 gene of lily peptide at 10 μg / ml and 100 μg / ml were 1.28 (p < 0.05) and 1.03 (p < 0.05), respectively.

[0126] Example 8: Anti-wrinkle and firming efficacy test of the lily flower peptide of the present invention

[0127] 1. Preparation of experimental reagents

[0128] 1) Complete culture medium: Add FBS to DMEM high-glucose culture medium to make the content 10%, double antibody 1%, and store at 4°C. The validity period is 30 days.

[0129] 2) Test samples: should be prepared on the day of the test, preferably within 4 hours before the test.

[0130] -Liquid samples: need to be filtered and sterilized, then diluted with cell culture medium to the corresponding concentration, generally starting from 10% and diluted to 6-12 concentration gradients;

[0131] - Water-soluble solid samples: first dissolve to a high concentration with complete culture medium, filter and sterilize, then dilute to the corresponding concentration with complete culture medium;

[0132] - Non-water-soluble samples: first dissolve to a high concentration with DMSO / anhydrous ethanol, then dilute to the corresponding concentration with complete culture medium. The highest concentration of the solvent in the final cytotoxicity experiment should not exceed 1%.

[0133] 3) Positive control: TGF-β 100 ng / mL.

[0134] 2. Test Method

[0135] 2.1 Cytotoxicity test

[0136] 1) Cell plating: discard the medium in the T75 culture flask, wash twice with PBS, add 1 mL of trypsin, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of fresh culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant. Add 1 mL of DMEM complete medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 pcs / mL were inoculated into a 96-well plate, 100 μL / well, and after inoculation, the plate was placed in a 37°C, 5% CO2 incubator for 24 h±2 h;

[0137] 2) Sample addition: Take out the 96-well plate, discard the supernatant, add complete medium to the normal control group, add complete medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 h±2 h;

[0138] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value; Cell viability calculation formula:

[0139] Cell viability (%) = OD value of sample group / OD value of normal control group × 100%

[0140] Subsequent efficacy evaluation experiments were conducted on samples with a safe concentration of more than 90% cell viability.

[0141] 2.2 Efficacy evaluation

[0142] 1) Cell plating: discard the culture medium in the T75 culture flask, wash twice with PBS, add 1 mL of trypsin, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant. Add 1 mL of complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated into a 96-well plate, 100uL / well, and placed in a 37°C, 5% CO2 incubator for 24h±2h after inoculation.

[0143] Note: A blank control group, a test sample group and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0144] 2) Sample addition: After 24 hours of culture, carefully discard the culture supernatant, add 150 μL of the sample of different concentrations to each well, and continue to culture for 24 hours ± 2 hours. Note: The blank control group contains only cells and complete culture medium, and the positive control group contains complete culture medium containing TGF-β100 ng / mL.

[0145] 3) Collection of cell supernatant: After the culture is completed, the cell culture supernatant is aspirated, centrifuged at 4000 rpm for 5 min to remove a small amount of floating cells, and the supernatant is collected.

[0146] 4) Detection: ELISA testing must be performed according to the instructions of the enzyme-linked immunosorbent assay kit. If the sample cannot be tested in time, the supernatant can be collected in a 1.5 mL sterile centrifuge tube and placed in a -20°C ultra-low temperature refrigerator for storage.

[0147] 3. Results and Statistics

[0148] 1) Calculation of protein content

[0149] Use professional curve making software to draw a standard curve with the concentration of the standard in the type I collagen detection kit as the ordinate and the corrected OD450 value of the standard as the abscissa to obtain the regression equation. Substitute the OD value of the test substance into the equation to obtain the protein content.

[0150] 2) Calculation of protein expression improvement rate

[0151] Protein expression enhancement rate % = (mean protein value of sample group / mean protein value of blank control group-1) × 100%

[0152] The experimental data were plotted using Graphpad Prism software, and the data between multiple groups were analyzed using one-way ANOVA, or the comparison between groups was analyzed using t-test statistics. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference.

[0153] Experimental Results

[0154] As can be seen from Table 15, compared with the blank group, positive TGF-β at 100ng / ml significantly promoted the secretion of elastin ELN1 (P<0.01), and had a firming effect; compared with the blank control, lily peptide increased the ELN1 protein content in a concentration-dependent manner within the concentration range of 10-40μg / ml, and had a firming effect; among them, at the concentrations of 10μg / ml and 40μg / ml, the effects were significant (P<0.05). L-carnosine significantly increased ELN1 protein in the concentration range of 20-40μg / ml (P<0.01 or 0.05); blue copper peptide did not significantly increase ELN1 protein in the concentration range of 10-40μg / ml; acetyl hexapeptide-8 significantly increased ELN1 protein in the concentration range of 20-40μg / ml (P<0.01 or 0.05); palmitoyl tripeptide-1 / 5 significantly increased ELN1 protein in the concentration range of 10μg / ml (P<0.05).

[0155] As shown in Table 16, compared with the blank control group, the positive control significantly promoted the secretion of type I collagen (p<0.001), and had anti-wrinkle effect; compared with the blank control, lily peptide had anti-wrinkle effect at concentrations of 10μg / ml and 100μg / ml, and could significantly promote the secretion of type I collagen, with the increase rates of 40.03% and 108.12% respectively. At concentrations of 10μg / ml and 20μg / ml, only palmitoyl tripeptide-1 / 7 could significantly promote the secretion of type I collagen, while L-carnosine, blue copper peptide, and acetyl hexapeptide-8 did not show anti-wrinkle effect.

[0156] Experiments have shown that lily flower peptide has anti-wrinkle and firming effects, and has certain advantages over existing peptide raw materials.

[0157] Table 15. Effects of samples on elastin production in HFF-1 cells

[0158]

[0159] Table 16. Effects of samples on type I collagen production in HFF-1 cells

[0160]

[0161] Example 9: Test of the repairing efficacy of the lily flower peptide of the present invention

[0162] 1. Cells

[0163] HFF-1 human dermal fibroblasts.

[0164] 2. Main reagents

[0165] DMEM medium (Gibco), fetal bovine serum (Gibco).

[0166] 3. Main equipment

[0167] CO2 incubator (healforce), biological safety cabinet (healforce), multifunctional microplate reader (Tecan).

[0168] 4. Experimental methods

[0169] 4.1 Cytotoxicity test of HFF-1 cells

[0170] 1) Inoculate cells in 96-well plates (1×10 4 / well) and incubated overnight in a 37°C, 5% CO2 incubator.

[0171] 2) Administration: Remove the culture medium, add 100 μL of culture medium to each well of the blank control group, and add 100 μL of culture medium containing the sample of corresponding concentration to each well of the sample group, and culture in a 37° C., 5% CO2 incubator for 24 h.

[0172] 3) Detection: Take out the 96-well plate, place it for about 10 minutes, equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, read the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula:

[0173] Cell viability (%) = OD value of sample group / OD value of blank control group × 100%

[0174] Note: Subsequent efficacy evaluation tests were performed when the cell viability was above 90%.

[0175] 4.2 HFF-1 human skin fibroblast I cell proliferation assay

[0176] 1) Inoculate cells in 96-well plates (1×10 4 / well) and incubated overnight in a 37°C, 5% CO2 incubator.

[0177] 2) Administration: Remove the culture medium, set up 3 replicates in each group, add 100 μL serum-free culture medium to each well of the blank control group (BC), add culture medium containing 10% fetal bovine serum to each well of the positive control group (PC), and add serum-free culture medium containing the test sample at the corresponding concentration to each well of the sample group. Culture for 24 hours.

[0178] 3) Detection: After 24 hours of culture, take out the 96-well plate, place it for about 10 minutes, equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, read the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula:

[0179] Cell viability (%) = OD value of sample group / OD value of blank control group × 100%

[0180] 4.3 Statistical analysis

[0181] The mean value (Mean) and standard deviation (SD) of each group of experiments were calculated, and Graphpad Prism software was used for data statistical analysis. P < 0.05 indicated a significant difference, P < 0.01 indicated an extremely significant difference, and P > 0.05 indicated no significant difference.

[0182] Experimental Results

[0183] As can be seen from Table 17, compared with the blank control group, the positive control significantly increased the viability of fibroblasts (p<0.01), indicating that the positive control test was effective; compared with the blank control, the fibroblast viability of lily flower peptide at concentrations of 20 and 100 μg / ml was 115.28% (P<0.05) and 117.68% (P<0.05), respectively, which can promote the proliferation of fibroblasts.

[0184] Carnosine can also significantly increase fibroblast viability at a concentration of 1-100μg / ml, which is equivalent to the effect of lily peptide. Acetyl hexapeptide-8, palmitoyl tripeptide-1 / tetrapeptide-7 can promote fibroblast viability at a concentration of 1μg / ml, but reduce cell viability at a concentration of ≥10μg / ml, showing cytotoxicity; blue copper peptide gradually reduces fibroblast viability at a concentration of 1-100μg / ml, and also shows certain cytotoxicity. Experiments show that the viability of fibroblasts treated with lily peptide samples is significantly improved, which can promote the proliferation of fibroblasts and has potential repair effects.

[0185] Table 17. Effects of samples on HFF-1 cell proliferation

[0186]

[0187] Example 10: Testing of the clock gene regulation effect of the lily flower peptide of the present invention

[0188] Reagent preparation

[0189] 1) Complete culture medium: Add FBS to DMEM (1×) high glucose medium to make the content 10%, double antibody 1%, and store at 4°C. The validity period is 30 days.

[0190] 2) Serum-free culture medium: Add 1% of double antibody to DMEM (1×) high-glucose culture medium and store at 4°C. The shelf life is 30 days.

[0191] 3) Test samples: The prepared samples are valid for 7 days.

[0192] -Liquid samples: need to be filtered and sterilized, then diluted with cell culture medium to the corresponding concentration, generally starting from 10% and diluted to 6-12 concentration gradients;

[0193] - Water-soluble solid samples: first dissolve to a high concentration in serum-free medium, filter and sterilize, then dilute to the corresponding concentration in serum-free medium;

[0194] - Water-insoluble samples: Dissolve to a high concentration with DMSO first, then dilute to the corresponding concentration with serum-free culture medium. Ensure that the DMSO concentration in the final culture medium does not exceed 1%.

[0195] Experimental methods

[0196] 1. Cytotoxicity test

[0197] 1) Cell plating: discard the culture medium in the T75 culture flask, wash twice with PBS, add 1ml of trypsin, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1ml of fresh culture medium to stop digestion, transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 4 minutes, and discard the supernatant. Add 1ml of DMEM complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 pcs / mL were inoculated into a 96-well plate, 100 μL / well, and after inoculation, the plate was placed in a 37°C, 5% CO2 incubator for 24 h±2 h overnight;

[0198] 2) Sample addition: Take out the 96-well plate, discard the supernatant, add serum-free culture medium to the normal control group, add serum-free DMEM high-glucose culture medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 h±2 h;

[0199] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value; Cell viability calculation formula:

[0200] Cell viability (%) = sample OD value / blank OD value × 100%

[0201] Sample concentrations with cell viability above 90% were subjected to subsequent efficacy evaluation tests.

[0202] 2. Efficacy evaluation

[0203] 1) Cell plating: discard the culture medium in the T75 culture flask, wash twice with PBS, add 1ml of trypsin, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1ml of fresh culture medium to stop digestion, transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 4 minutes, and discard the supernatant. Add 1ml of complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated with 2 mL / well in a 6-well plate. After inoculation, the plate was placed in a 37°C, 5% CO2 incubator for 24 h ± 2 h.

[0204] 2) Sample addition: After culturing for 24 hours, carefully discard the culture supernatant, add 2 mL of the sample of different concentrations to each well, and continue culturing for 24 hours ± 2 hours.

[0205] 3) Sample collection: After the culture is completed, the culture supernatant is discarded, the cells are washed three times with PBS, and then 1 ml of RNA extraction reagent is added to each well;

[0206] 1. RNA extraction steps:

[0207] ① Add chloroform (1 / 5 volume of RNAiso Plus) to the homogenate lysate in the above step, cover the centrifuge tube tightly, and mix until the solution is emulsified and milky white;

[0208] ②Let stand at room temperature for 5 minutes.

[0209] ③ Centrifuge at 12,000×g for 15 minutes at 4℃. Carefully remove the centrifuge tube from the centrifuge. The homogenate is now divided into three layers: a colorless supernatant (containing RNA), a white protein layer in the middle (mostly DNA), and a colored lower organic phase.

[0210] ④ Aspirate the supernatant and transfer it to another new centrifuge tube (do not aspirate the white middle layer).

[0211] ⑤ Add 0.5-1 times the volume of RNAiso Plus isopropanol to the supernatant, invert the tube to mix thoroughly, and let stand at room temperature for 10 minutes.

[0212] ⑥ Centrifuge at 12,000×g for 10 minutes at 4℃. Generally, RNA precipitate will appear at the bottom of the test tube after centrifugation.

[0213] 2. RNA precipitation cleaning: Carefully discard the supernatant and do not touch the precipitate. It does not matter if there is a small amount of isopropanol left. Add 75% ethanol equal to the amount of RNAiso Plus, gently invert the centrifuge tube to wash the wall, centrifuge at 7,500×g and 4℃ for 5 minutes, then carefully discard the supernatant and do not touch the precipitate.

[0214] 3. RNA dissolution: Open the centrifuge tube cap and dry the precipitate at room temperature for a few minutes. After the precipitate is dry, add an appropriate amount of RNase-free water to dissolve the precipitate. NOTE: Do not centrifuge or heat dry, otherwise the RNA will be difficult to dissolve.

[0215] 4. RNA Concentration Determination

[0216] Nucleic acid quantification was performed on an ELISA reader to calculate the RNA concentration of each sample.

[0217] Reverse transcription cDNA step: Use a reverse transcription kit to synthesize the first-strand cDNA and establish a 20 μL reaction system with 500 ng-1 μg total RNA.

[0218] Reaction for removing genomic DNA: Prepare the reaction mixture on ice according to the ingredients shown in Table 18. To ensure the accuracy of the reaction mixture, prepare the Master Mix according to the amount of reaction number + 2 for each reaction, then dispense it into each reaction tube, and finally add the RNA sample. 42℃2min (or room temperature 5min*2)

[0219] Table 18

[0220] Reagents Usage 5×gDNA Eraser Buffer 2μL gDNA Eraser 1μL Total RNA - RNase-Free ddH2O Make up to 10 μL

[0221] In the 20μL reverse transcription reaction system shown in Table 19, TB Green qPCR method can use up to 1μg of TotalRNA, and the reaction time can be extended to 30 minutes at room temperature. Prepare the mixed solution ix according to the reverse transcription system in Table 2, add it to the reaction solution in the gDNA removal step, and mix it thoroughly. Incubate at 37℃ for 15min, incubate at 85℃ for 5s, and then put it on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

[0222] Table 19

[0223] Reagents Usage Reaction solution of step 1 10μL PrimeScript RT Enzyme Mix I 1μL RT Primer Mix*4 1μL 5×Prime Script Buffer 2(for Real Time) 4μL RNase-Free ddH2O 4μL Total 20μL

[0224] The M in the reverse transcription reaction was used for real-time fluorescence quantitative PCR; the upstream and downstream primers β-actin, PER, CLOCK, and BAMLI were prepared into 100μM master solution with RNase-Free ddH2O, and then the upstream and downstream were mixed and diluted 1:1 to 10μM working solution and placed on ice. The cDNA obtained by reverse transcription was diluted 3 times for subsequent experiments. According to Table 20, a 20μL reaction system was established for qPCR reaction.

[0225] Table 20

[0226] Composition Usage TB Green Premix Ex Taq(Tli RNase H Plus)(2X) 10μL Primers 0.8μL RNase-Free ddH2O 6.8μL ROX Reference Dye II(50X)*2 0.4μL cDNA 2μL Total 20μL

[0227] Perform Real Time PCR reaction: It is recommended to use the two-step PCR reaction procedure shown in the following diagram. If the procedure does not produce good experimental results, optimize the PCR conditions. If the amplification performance of the two-step PCR reaction is poor due to the use of primers with lower Tm values, you can try the three-step PCR amplification reaction.

[0228] According to the C(t) of β-actin, PER, CLOCK and BAMLI in each experimental group, the relative expression of RNA of PER, CLOCK and BAMLI in each concentration group was calculated as follows:

[0229] ΔC(t)=C( t) 目的基 因 -C(t) β-actin

[0230] ΔΔC(t)=ΔC(t) 空白对照组 -ΔC(t) 受试物测试组

[0231] Relative RNA expression = 2^ΔΔC(t)

[0232] If a co-solvent is used in the preparation of the test substance, the blank control group value in the formula is replaced by the solvent control group value.

[0233] Statistical analysis: The mean value (Mean) and standard deviation (Standard Deviation, SD) of each group of experiments were calculated. The statistical processing results were expressed as Mean ± SD. GraphPad Prism software was used to perform variance analysis on the data, and the blank control group (or solvent control group) was used as the standard to compare the relative expression of the target gene in each experimental group. P < 0.05 indicated a significant difference.

[0234] Experimental Results

[0235] like Figures 2 to 4The results showed that 0.5 mg / ml of lily peptide could significantly upregulate the expression of Bmal1 gene (P<0.001) and downregulate the expression of CLOCK gene and Per gene (P<0.01), thus having the function of regulating clock genes.

[0236] Example 11: Photoprotective effect test of the lily flower peptide of the present invention

[0237] Materials and methods

[0238] 1. Cells: HaCaT keratinocytes.

[0239] 2. Main reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), CCK8 kit.

[0240] 3. Main equipment: CO2 incubator (healforce), biological safety cabinet (healforce), multifunctional microplate reader (Tecan), photoaging model UV equipment (Vilber Bio-Sun).

[0241] 4. Experimental methods

[0242] 1) Plate: Inoculate cells in 96-well plates (2×10 5 / well) and incubated overnight in a 37°C, 5% CO2 incubator.

[0243] 2) Modeling: When the relative cell density of the cells reached 70%, the culture medium was removed, 100 μL PBS was added to cover the cells, and then the cells were placed directly under the UVB lamp. The PBS was discarded when the cumulative radiation dose reached 30 mJ / cm2.

[0244] 3) Administration: The blank control group (BC) was added with serum-free culture medium, the model group was added with serum-free culture medium, and the test sample group was added with drug-containing serum-free culture medium, and the culture was continued for 24 hours.

[0245] 3) Detection: After 24 hours of culture, take out the 96-well plate, place it for about 10 minutes, equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, read the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula:

[0246] Cell viability (%) = OD value of sample group / OD value of blank control group × 100%

[0247] 5. Statistical analysis

[0248] The mean value (Mean) and standard deviation (SD) of each group of experiments were calculated, and Graphpad Prism software was used for data statistical analysis. P < 0.05 was considered a significant difference, P < 0.01 was considered an extremely significant difference, and P > 0.05 was considered to be insignificant.

[0249] Experimental Results

[0250] Test results: See Table 21. As can be seen from Table 21, compared with the blank control group, the cell viability of the model group was significantly reduced (p<0.001), and the experiment was effective; compared with the model group, at 20 and 100 μg / ml concentrations, the cell viability of the lily peptide group was 154.36% (p<0.01) and 58.71% (p<0.01), respectively, which can reduce UVB damage to cells and has a photoprotective effect; L-carnosine, blue copper peptide, acetyl hexapeptide-8, palmitoyl tripeptide-1 / 7 and palmitoyl tripeptide-5, all had no photoprotective effect on cells.

[0251] Table 21. Effects of samples on the viability of UVB-damaged keratinocytes

[0252]

[0253] The enzyme-linked immunosorbent assay (ELISA) was used to test the content of matrix metalloproteinase MMP-1 in the cell supernatant, and the effects of the samples on the level of MMP-1 in photodamaged keratinocytes were calculated to evaluate the protective effects of the tested samples on photodamaged cells.

[0254] Materials and methods

[0255] 1. Cells: HaCat keratinocytes.

[0256] 2. Main reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), L-ascorbic acid (McKinlin), ELISA kit (Huamei Biotechnology).

[0257] 3. Main equipment: CO2 incubator (healforce), biological safety cabinet (healforce), multifunctional microplate reader (Tecan), photoaging model UV equipment (Vilber Bio-Sun).

[0258] 4. Experimental methods

[0259] 1) Plate: Inoculate cells in 96-well plates (2×10 5 / well) and incubated overnight in a 37°C, 5% CO2 incubator.

[0260] 2) Modeling: When the relative cell density of the cells reached 70%, the culture medium was removed, 100 μL PBS was added to cover the cells, and then the cells were placed directly under the UVB lamp. The PBS was discarded when the cumulative radiation dose reached 30 mJ / cm2.

[0261] 3) Administration: Complete culture medium was added to the blank control group and the model group, 100 μg / mL VC was added to the positive control group, and drug-containing complete culture medium was added to the test sample group, and the culture was continued for 24 h.

[0262] 3) Detection: After 24 hours of culture, the supernatant was centrifuged and then detected and calculated using an enzyme-linked immunosorbent assay kit.

[0263] 4) Statistical analysis: The mean (Mean) and standard deviation (SD) of each group of experiments were calculated, and Graphpad Prism software was used for data statistical analysis. P < 0.05 was considered a significant difference, P < 0.01 was considered an extremely significant difference, and P > 0.05 was considered to be insignificant.

[0264] Experimental Results

[0265] As shown in Table 22, compared with the blank control group, the model group significantly increased the MMP-1 content (p<0.001), and the experiment was effective; compared with the model group, at a concentration of 100 μg / ml, lily peptide can significantly reduce the MMP-1 content, which is better than VC and carnosine at the same concentration; at a concentration of 10 μg / ml, lily peptide can still significantly reduce the MMP-1 content, which is better than carnosine, blue copper peptide, palmitoyl tripeptide-1 / 7 and acetyl hexapeptide-8 at the same concentration. The experiment proved that lily peptide has a photoprotective effect, which can resist UVB-induced increase in MMP-1 levels and reduce light damage.

[0266] Table 22. Effects of samples on the expression level of MMP-1 in keratinocytes stimulated by UVB

[0267]

[0268]

[0269] Example 12: Anti-photodamage effect of the lily flower peptide of the present invention

[0270] The UVA-damaged human fibroblast model was used to investigate the effects of the samples on the viability of photodamaged cells, and the anti-photodamage efficacy of the samples was evaluated in vitro.

[0271] Materials and methods

[0272] 1. Cells: Human skin fibroblasts, Nanjing Kebai Biotechnology Co., Ltd.

[0273] 2. Main reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), etc.

[0274] 3. Main equipment: CO2 incubator (healforce), biological safety cabinet (healforce), multifunctional microplate reader (Tecan), photoaging model UV equipment (Vilber Bio-Sun), etc.

[0275] 4. Experimental methods

[0276] 1) Cell plating: discard the medium in the T25 culture flask, wash twice with PBS, add 1 mL of 0.25% trypsin, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant. After resuspending with 1 mL of complete culture medium, count the cells. Dilute the cell suspension to a density of 8×10 4 Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0277] Note: A blank control group, a sample group and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0278] 2) Sample addition: Carefully discard the culture supernatant and set up the following groups: ① Blank control group: 100 μL complete culture medium; ② Positive control group: 100 μL 100 μg / mL VC; ③ Sample group: add samples of different concentrations.

[0279] 3) Modeling: UVA 60J / cm2 was selected according to the request of the sender, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours after irradiation.

[0280] 4) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula: Cell viability (%) = OD value of sample group / OD value of blank control group × 100%

[0281] Experimental Results

[0282] As shown in Table 23, compared with the normal control group, the cell viability decreased by nearly 30% (P<0.05), indicating that UVA has caused significant light damage to the cells; compared with the model group, 10μg / mL and 100μg / mL of lily polypeptide can significantly improve cell viability (P<0.05), indicating that it has an anti-light damage effect. 10μg / mL tripeptide-32 can also significantly protect cells, but the overall effect is not as good as lily polypeptide. VA derivative-HPR not only did not improve cell viability, but further aggravated cell light damage.

[0283] Table 23. Effects of samples on the viability of UVA-damaged fibroblasts

[0284]

[0285]

[0286] Example 13: Test on the moisturizing effect of the lily flower peptide of the present invention

[0287] To investigate the effect of samples on the expression of water channel protein (AQP3) in skin keratinocytes and to evaluate cosmetic raw materials / products with potential moisturizing effects in vitro.

[0288] Materials and Methods

[0289] 1. Cells: Human keratinocytes (HaCaT), Zhejiang Meisen Cell Technology Co., Ltd. CTCC-002-0012.

[0290] 2. Instruments: electronic balance ME204E, Mettler; CO2 incubator HF90, Healforce; biological safety cabinet 1500LC, Healforce; inverted microscope CKX53, OLYMPUS; centrifuge L3-5K, Eppendorf; cell counter Countess3, Thermo Fisher; multifunctional microplate reader EPLEX, Tecan; refrigerator YCD-EL300, Meiling; constant temperature water bath HWS-24, Shanghai Yiheng; adjustable pipette 10 / 100 / 200 / 1000 / 5000ul, Eppendorf.

[0291] 3. Experimental reagents and consumables: DMEM high-glucose medium, Gibco; fetal bovine serum (FBS), Gibco; trypsin, Biosharp / Gibco; PBS phosphate buffer, Solebao; penicillin-streptomycin mixture (double antibody), ZetaLife; culture flask, BIOFIL; 96-well plate, Afiy; 1.5 mL centrifuge tube, Afiy; dimethyl sulfoxide cell culture grade (DMSO), Solebao; glycerol, bulk; CCK8, ZetaLife; AQP3 Elisa Kits, Shanghai ELISA.

[0292] 4. Experimental methods

[0293] 1) Cell plating: discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off under a microscope, add 5 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 1 mL of complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 100 μL / well was inoculated into a 96-well plate, and the plate was incubated in a 37°C, 5% CO2 incubator overnight. Note: A blank control group, a test sample group, and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0294] 2) Sample addition: After 24 hours of culture, the cell culture medium was replaced with the culture medium containing the test sample, 200 μL / well, and culture was continued for 24 hours. The positive control group was a complete culture medium containing 3% glycerol.

[0295] 3) Detection: Place the 96-well plate at 4°C, centrifuge at 1000g for 10 minutes, take 100μL of the supernatant from each well and place it on a new 96-well plate for immediate detection (or freeze it in a -20°C refrigerator and test it within one week). Prepare the required standards, antibodies, horseradish peroxidase-labeled avidin working solution, colorimetric solution and stop solution, etc.; dilute the sample according to the protein detection range of the kit to make it suitable for the standard curve, centrifuge at 1000g for 10 minutes, add it to the ELISA plate, add HRP 100ul / well at the same time, and incubate it in a 37°C incubator for 60 minutes; discard the supernatant, wash it 5 times with the prepared washing solution; add staining solution A and B for color development for 15min to 25min; finally, add the stop solution, detect the absorbance value at 450nm with an ELISA reader within 15min, and calculate the AQP3 protein expression level.

[0296] 4) Data analysis and statistics: The calculation formula is as follows: AQP3 up-regulation rate % = (AQP3 content in the experimental group - AQP3 content in the blank control group) / (AQP3 content in the blank control group) × 100%. The experimental data were plotted using Graphpad Prism software, and the data between multiple groups were analyzed using one-way ANOVA, or the comparison between each group was analyzed using t-test statistical analysis. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0297] Experimental Results

[0298] As shown in Table 24, compared with the blank control group, 0.5% lily peptide can significantly increase the cell AQP-3 content (P<0.05), with an upregulation rate of 27.2%, and has a moisturizing effect.

[0299] Table 24. Effects of samples on AQP-3 protein expression in Hacat cells

[0300]

[0301] Example 14: Safety of the Lily Flower Peptide of the Present Invention

[0302] Materials and methods:

[0303] 1 Experimental materials: SPF-grade fertilized chicken embryos of Bailaihang chicken, sodium chloride (Solarbio), sodium hydroxide (Shanghai McLean Co., Ltd.)

[0304] 2 Experimental method: Based on SN / T2329-2009 "Chicken embryo chorioallantoic membrane test for eye irritation / corrosion of cosmetics"

[0305] Using the endpoint evaluation method, 0.3 ml of the sample was dripped onto the CAM. After 3 minutes of action, the test substance on the CAM membrane was gently rinsed with normal saline to observe the bleeding, coagulation, and vascular dissolution, and photos were taken. At the same time, a negative control of 0.9% NaCl and positive controls of 0.2%, 0.3%, and 0.5% sodium hydroxide solutions were set.

[0306] The results are as follows Figures 5-6 As shown, the sample was tested by the endpoint evaluation method. After 3 minutes of the sample action: compared with the negative control group, no obvious bleeding, coagulation, and vascular fusion were observed in the CAM at the concentrations of 10 mg / ml and 5 mg / ml of lily peptide, so the ES score of the sample lily peptide at the concentrations of 10 mg / ml and 5 mg / ml was 0. The experiment showed that the lily peptide was non-irritating at the concentrations of 10 mg / ml and 5 mg / ml.

[0307] Example 15: Human skin care efficacy of the lily flower peptide of the present invention

[0308] Purpose of the test: Under normal circumstances, after adult subjects use the test samples continuously for 28 days according to the instructions, evaluate whether the test samples have anti-wrinkle, firming (immediate & long-term), repair, moisturizing, and soothing effects.

[0309] Test samples: Group 1: Lily flower peptide essence AVS on the left and Lily flower peptide essence A on the right; Group 2: Lily flower peptide essence B on the left VS Lily flower peptide essence B on the right; Note: Left A and left B represent base; Right A represents 0.1%; Right B represents 0; Final effective number of people: ≥10 people per group.

[0310] Inclusion criteria of subjects

[0311] 1. Women aged between 30 and 45 are preferred;

[0312] 2. Dry skin or combination skin;

[0313] 3. The sensitive skin questionnaire screened the sensitive skin population (subjects with a high transepidermal water loss rate, with a value of >15g / m2h being the best);

[0314] 4. The wrinkle level at the corner of the eye is level 1 or above (wrinkle level 2-4 is the best);

[0315] 5. People who think their skin is loose;

[0316] 6. People who think their skin easily turns red.

[0317] Exclusion criteria for subjects

[0318] 1. Those who have tried antihistamines in the past week or immunosuppressants in the past month;

[0319] 2. Those who have used any anti-inflammatory drugs in the tested area within the past two months;

[0320] 3. Patients with clinically uncured inflammatory skin diseases;

[0321] 4. Patients with insulin-dependent diabetes;

[0322] 5. Patients who are receiving treatment for asthma or other chronic respiratory diseases;

[0323] 6. Those who have received anti-cancer chemotherapy in the past 6 months;

[0324] 7. Patients with immunodeficiency or autoimmune diseases;

[0325] 8. Lactating or pregnant women;

[0326] 9. Patients who have undergone bilateral mastectomy and bilateral axillary lymph node removal;

[0327] 10. Judges whose test results may be affected by scars, pigmentation, atrophy, port wine stains or other blemishes on the skin to be tested;

[0328] 11. Investigators currently participating in other clinical trials.

[0329] Product application area: face;

[0330] How to use: It is recommended to use this product once a day, morning and evening. Each time you use it, please squeeze out 1-2 pumps of the product and apply it evenly on one side of the face. (Use Lily Peptide Essence Left A / Lily Peptide Essence Left B on the left side of the face, and use Lily Peptide Essence Right Left A / Lily Peptide Essence Right B on the right side of the face)

[0331] Test cycle: D0 (before use), D14 (after use), D28 (after use);

[0332] Test Notes:

[0333] 1) Before the test, the subjects need to sit quietly in a standard test environment for at least 30 minutes without drinking water or beverages.

[0334] 2) Do not wear a mask during meditation, stay relaxed, and avoid touching the tested area.

[0335] Test items:

[0336] 1. Environmental conditions: (21±2)℃, (50±10)%RH;

[0337] 2. Instrument measurement

[0338] 2.1 Anti-wrinkle: wrinkle parameters, testing instrument: VISIA; testing site: outer canthus; time points: D0 (before use), Timm (15 minutes after using the sample), D14 (after use), D28 (after use);

[0339] 2.2 Firming: R2, F4; Testing instrument: Cutometer; Testing site: Cheek; Time point: D0 (before use), D14 (after use), D28 (after use);

[0340] 2.3 Repair: transepidermal water loss, moisture content, a value; Testing instruments: Tewameter, Corneometer, Visia; Testing site: cheek; Time points: D0 (before use), D14 (after use), D28 (after use);

[0341] 2.4 Soothing: a value; Testing instrument: Visia; Testing site: Cheek; Time point: D0 (before use), D14 (after use), D28 (after use);

[0342] 2.5 Moisturizing: moisture content, testing instrument: Corneometer; testing site: cheek; time points: D0 (before use), D14 (after use), D28 (after use);

[0343] 2.5 Fill in the questionnaire

[0344] Testing process:

[0345] 1. Recruit test volunteers according to the enrollment requirements and sign written informed consent and other relevant forms;

[0346] 2. Fill out the sensitive skin screening questionnaire online or offline, and proceed to the next step if you meet the requirements;

[0347] 3. After cleaning with unified cleansing products, sit quietly for at least 30 minutes, fill in the self-evaluation questionnaire during this period, and measure the initial value of D0 with visia, water loss probe, water content probe, and elasticity probe;

[0348] 4.Distribute samples and explain product usage and requirements to subjects;

[0349] 5. After using the sample for 15 minutes, take a photo of the visit;

[0350] 6. On the 14th day after using the sample, fill in the self-assessment questionnaire and measure the D14 value using visia, moisture loss probe, moisture content probe, and elasticity probe;

[0351] 7. On the 28th day after using the sample, fill in the self-assessment questionnaire and measure the D28 value using visia, moisture loss probe, moisture content probe, and elasticity probe.

[0352] Test results: See Table 25 and Table 26.

[0353] Table 25. Instrument test results

[0354]

[0355] Table 26. Questionnaire self-assessment results

[0356]

[0357]

[0358] Note: The questionnaire has 9 points. The higher the score, the better the skin condition.

[0359] Test conclusion: Lily flower peptide has soothing / redness removal, firming effects, and certain repair / moisturizing potential; among them, 0.5% essence is better than 0.1% essence.

[0360] Example 16: Safety of the Lily Flower Peptide of the Present Invention on Human Body

[0361] Safety test: Human skin closed patch test

[0362] Method: Human skin patch test method-Cosmetic Safety Technical Specifications 2015 Edition;

[0363] Test results: The results showed that 3 out of 30 people who took the blank matrix sample of lily flower peptide essence had grade 1 adverse reactions within 0.5 h. When the essence was added at 0.02%, 0.1% and 0.5%, 2, 3 and 1 people had grade 1 adverse skin reactions respectively. It was safe and non-irritating, as shown in Table 27.

[0364] Table 27. Human patch test results

[0365]

Claims

1. A method for preparing lily peptide, comprising the following steps: S1. Crush, dissolve and filter the lily raw material to obtain a filtrate; S2, adjusting the pH value of the filtrate until the precipitate is completely precipitated and filtering to obtain the precipitate; S3, adding a composite enzyme to the precipitate for enzymolysis to obtain an enzymolysis product, wherein the composite enzyme comprises flavor protease and papain; S4, fermenting the enzymatic hydrolysis product using bifidobacteria; and S5. Obtain lily flower peptide.

2. The preparation method according to claim 1, characterized in that: In step S3, the concentration ratio of flavor protease:papain is (1-5):(5-1), preferably, the concentration ratio of flavor protease:papain is 1:

1.

3. The preparation method according to claim 1, characterized in that: In step S3, the concentration of the complex enzyme is 5000-20000 U / g, preferably 8000-15000 U / g, and more preferably 12000 U / g.

4. The preparation method according to claim 1, characterized in that: In step S3, the conditions for the enzymatic hydrolysis are a temperature of 50 to 60° C., a pH of 7 to 9, and a reaction time of 3 to 10 hours.

5. The preparation method according to claim 1, characterized in that: In the step S4, the bifidobacterium is one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum, or Bifidobacterium thermophilum.

6. The preparation method according to claim 1, characterized in that: The prepared lily flower peptide includes a polypeptide mixture as shown below: DQLSDLQR,VEIVDDIHAAIDHIHQ,ADTGGFLEV,MIIAVPTG,NFEGSIP,LLQDIVPTF,CIQGLPEG,AVEEAGGPK,PEDIAPNK,NLTDDDVV,S DEGDVLY, QKGDQYLNS, QAAASKAATMD, SAEAIVLIGTA, GIAVLLK, INEEAGDEIKEAS, SNVGGSGAVQSV, EKGGVFFC, DPSSFPP, WSPSMIK.

7. A lily flower peptide obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the lily flower peptide according to claim 7 for preparing skin care products.

9. A skin care product with multiple functions, comprising the lily flower peptide as claimed in claim 7.

10. The product according to claim 9, characterized in that The skin care product has multiple functions, including one or more of regulating clock genes, moisturizing, soothing, repairing, anti-wrinkle, firming, repairing, photoprotection, or anti-photodamage.

Images