Freeze-dried powder and preparation method thereof

Abstract

The invention belongs to the field of cosmetics, and discloses freeze-dried powder and a preparation method thereof.The freeze-dried powder is prepared from, by weight, 0-10 parts of polysaccharide, 0-3 parts of hyaluronic acid, 0.0001-1.5 parts of polypeptide active matter, 0.01-1.5 parts of protein, 0.1-0.7 part of buffering agent and 2-10 parts of excipient. All the raw materials except the excipient are functional components, and the final product obtained by combining a freeze-drying process is more suitable for realizing unobstructed absorption of the functional components of human skin; the freeze-dried powder disclosed by the invention has the effects of repairing, tightening and resisting wrinkles.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics, and particularly relates to a freeze-dried powder and a preparation method thereof. Background Art

[0002] As researchers continue to explore the secrets of human aging, the perspective of biological aesthetics will shift from the macroscopic level to the regulation and sublimation of cells and tissues at the microscopic level. Cells, as the main basic functional units of the human body, are the basis for the occurrence, development and aging of the skin. The health of skin cells depends on internal metabolism and external nutrient supply. The biological activity of peptide actives can promote cell proliferation and differentiation, repair damaged cells, and are therefore widely used in beauty and skin care products and pharmaceuticals. At present, it is still necessary to develop a product that meets the unique market needs of high efficacy, high purity, and easy absorption. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0004] On the one hand, the present invention provides a freeze-dried powder, comprising the following raw materials in parts by weight: 0 to 10 parts of polysaccharide, 0 to 3 parts of hyaluronic acid, 0.0001 to 1.5 parts of polypeptide active ingredients, 0.01 to 1.5 parts of protein, 0.1 to 0.7 parts of buffer, and 2 to 10 parts of excipient.

[0005] Preferably, the polysaccharide comprises at least one of Tremella fuciformis heteropolysaccharide, soluble proteoglycan, trehalose and Tremella fuciformis polysaccharide.

[0006] Preferably, the hyaluronic acid comprises at least one of hydrolyzed hyaluronic acid and sodium hyaluronate.

[0007] Preferably, the polypeptide active ingredient includes at least one of oligopeptide, oligopeptide-3, oligopeptide-2, oligopeptide-4, snake venom peptide, blue copper peptide, acetyl hexapeptide-8, carnosine, pentapeptide-3 and tripeptide-1.

[0008] Preferably, the protein includes at least one of hydrolyzed collagen, type I recombinant collagen, type II recombinant collagen, type III recombinant collagen, fibronectin and serum albumin.

[0009] Preferably, the buffer comprises at least one of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0010] Preferably, the excipient comprises mannitol.

[0011] Preferably, the freeze-dried powder of the present invention may also include HP skin wrinkle smoothing agent and Bossagra.

[0012] On the other hand, the present invention also provides a method for preparing the above-mentioned freeze-dried powder, comprising the following steps:

[0013] The polysaccharide, the buffer, the excipient and water are mixed, filtered and sterilized to obtain phase A;

[0014] Mix the hyaluronic acid and water, and filter to obtain phase B;

[0015] Taking part of the phase A, mixing it with the polypeptide active substance and the protein, filtering it to obtain phase C, and mixing the phase C, the remaining phase A and the phase B to obtain a lyophilized powder liquid;

[0016] The freeze-dried powder liquid is freeze-dried to obtain the freeze-dried powder.

[0017] Preferably, the filtration is performed using a 0.20-0.50 μm filter.

[0018] Preferably, the freeze-drying process comprises the following steps:

[0019] Pre-freezing treatment, wherein the temperature of the pre-freezing treatment is -35 to 55°C and the time of the pre-freezing treatment is 2 to 3 hours;

[0020] Vacuum treatment;

[0021] Sublimation treatment, the sublimation treatment includes a first sublimation treatment, a second sublimation treatment and a third sublimation treatment in sequence, the temperature of the first sublimation treatment is -15 to 25°C, the time of the first sublimation treatment is 8 to 10 hours, the temperature of the second sublimation treatment is -3 to 8°C, the time of the second sublimation treatment is 4 to 6 hours, the temperature of the third sublimation treatment is 0°C, and the time of the third sublimation treatment is 0.5 to 1.5 hours;

[0022] Temperature treatment, wherein the temperature treatment includes a first temperature treatment, a second temperature treatment and a third temperature treatment in sequence, the temperature of the first temperature treatment is 4 to 6°C, the temperature of the second temperature treatment is 13 to 17°C, the temperature of the third temperature treatment is 28 to 33°C, and the time of the first temperature treatment, the second temperature treatment and the third temperature treatment are all 1 to 2.5 hours;

[0023] The heat preservation treatment is performed at a temperature of 25 to 45° C. and a time of 5 to 9 hours.

[0024] The beneficial effects of the present invention are as follows: (1) Except for the excipient, all the raw materials of the present invention are effective ingredients. The final product obtained by combining the freeze-drying process is more suitable for the unimpeded absorption of the effective ingredients of human skin; (2) The polypeptide active substance and protein in the raw materials of the present invention have independent absorption mechanisms in the human body and do not interfere with each other, which helps to reduce the absorption inhibition caused by the competition between the two components for the common absorption sites; (3) The freeze-dried powder of the present invention has the effects of repairing, firming and anti-wrinkle. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The product soothing evaluation results of the freeze-dried powder of Example 1 of the present invention;

[0026] Figure 2 This is a picture of the red area on the face of a subject in the evaluation of the freeze-dried powder of Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] Example 1

[0029] A freeze-dried powder comprises the following raw materials in parts by weight: 1 part of trehalose, 0.01 part of oligopeptide-2, 0.01 part of oligopeptide, 0.5 part of serum albumin, 0.437 part of disodium hydrogen phosphate, 0.12 part of sodium dihydrogen phosphate and 6 parts of mannitol.

[0030] The freeze-dried powder preparation method of Example 1 is as follows:

[0031] 1 part by weight of trehalose, 0.437 parts by weight of disodium hydrogen phosphate, 0.12 parts by weight of sodium dihydrogen phosphate, 6 parts by weight of mannitol and 80 parts by weight of purified water were mixed, filtered, sterilized at 120° C. for 30 min to obtain phase A, and phase A was allowed to stand and cool to 38° C. for later use;

[0032] Take one third of phase A and mix with 0.01 weight part of oligopeptide-2, 0.01 weight part of oligopeptide, and 0.5 weight part of serum albumin, filter to obtain phase C, mix phase C and the remaining two thirds of phase A to obtain a lyophilized powder liquid, and dispense the lyophilized powder liquid into lyophilized powder bottles, with a canning volume of 1.6 mL / tube;

[0033] The freeze-dried powder liquid is freeze-dried to obtain freeze-dried powder.

[0034] The filtration during the preparation of freeze-dried powder in Example 1 was performed using a 0.20 μm filter device.

[0035] In the freeze-dried powder preparation process of Example 1, the freeze-drying process comprises the following steps:

[0036] Pre-freezing treatment: the temperature of the pre-freezing treatment is -35°C and the time of the pre-freezing treatment is 2h;

[0037] Vacuum treatment, the setting value of vacuum treatment is 0.5mbar, and the vacuum treatment time is 1h;

[0038] Sublimation treatment, the sublimation treatment includes a first sublimation treatment, a second sublimation treatment and a third sublimation treatment in sequence, the temperature of the first sublimation treatment is -15°C, the time of the first sublimation treatment is 8 hours, the temperature of the second sublimation treatment is -3°C, the time of the second sublimation treatment is 4 hours, the temperature of the third sublimation treatment is 0°C, and the time of the third sublimation treatment is 0.5 hours;

[0039] The heating treatment includes a first heating treatment, a second heating treatment and a third heating treatment in sequence. The temperature of the first heating treatment is 4°C, the temperature of the second heating treatment is 13°C, the temperature of the third heating treatment is 28°C, and the time of the first heating treatment, the second heating treatment and the third heating treatment is 1 hour;

[0040] The insulation treatment temperature is 25°C and the insulation treatment time is 5h.

[0041] Example 2

[0042] A freeze-dried powder comprises the following raw materials in parts by weight: 1 part of trehalose, 0.03 parts of sodium hyaluronate, 0.001 parts of acetyl hexapeptide-8, 0.001 parts of pentapeptide-3, 0.001 parts of tripeptide-1, 0.001 parts of oligopeptide, 0.001 parts of fibronectin, 0.02 parts of soluble collagen and 4 parts of mannitol.

[0043] The freeze-dried powder preparation method of Example 2 is as follows:

[0044] 1 part by weight of trehalose, 6 parts by weight of mannitol and 80 parts by weight of purified water were mixed, filtered, sterilized at 120° C. for 30 min to obtain phase A, and phase A was allowed to stand and cool to 38° C. for later use;

[0045] Mix 0.03 parts by weight of sodium hyaluronate and 15 parts by weight of purified water, and filter to obtain phase B;

[0046] Take one third of phase A and mix with 0.001 parts of acetyl hexapeptide-8, 0.001 parts of pentapeptide-3, 0.001 parts of tripeptide-1, 0.001 parts of oligopeptide, 0.001 parts of fibronectin, and 0.02 parts by weight of soluble collagen, filter to obtain phase C, mix phase C, the remaining two thirds of phase A and phase B to obtain a lyophilized powder liquid, and dispense the lyophilized powder liquid into lyophilized powder bottles, with a canning volume of 1.6 mL / tube;

[0047] The freeze-dried powder liquid is freeze-dried to obtain freeze-dried powder.

[0048] In the preparation process of freeze-dried powder in Example 2, filtration was performed using a 0.20 μm filter device.

[0049] In the freeze-dried powder preparation process of Example 2, the freeze-drying process comprises the following steps:

[0050] Pre-freezing treatment: the temperature of the pre-freezing treatment is -35°C and the time of the pre-freezing treatment is 2h;

[0051] Vacuum treatment, the setting value of vacuum treatment is 0.5mbar, and the vacuum treatment time is 1h;

[0052] Sublimation treatment, the sublimation treatment includes a first sublimation treatment, a second sublimation treatment and a third sublimation treatment in sequence, the temperature of the first sublimation treatment is -15°C, the time of the first sublimation treatment is 8 hours, the temperature of the second sublimation treatment is -3°C, the time of the second sublimation treatment is 4 hours, the temperature of the third sublimation treatment is 0°C, and the time of the third sublimation treatment is 0.5 hours;

[0053] The heating treatment includes a first heating treatment, a second heating treatment and a third heating treatment in sequence. The temperature of the first heating treatment is 4°C, the temperature of the second heating treatment is 13°C, the temperature of the third heating treatment is 28°C, and the time of the first heating treatment, the second heating treatment and the third heating treatment is 1 hour;

[0054] The insulation treatment temperature is 25°C and the insulation treatment time is 5h.

[0055] Example 3

[0056] A freeze-dried powder comprises the following raw materials in parts by weight: 0.002 parts of oligopeptide, 1 part of carnosine, 0.3 parts of blue copper peptide, 0.001 parts of fibronectin, 0.02 parts of soluble collagen, 0.437 parts of disodium hydrogen phosphate, 0.12 parts of sodium dihydrogen phosphate and 5 parts of mannitol.

[0057] The freeze-dried powder preparation method of Example 3 is as follows:

[0058] 0.437 parts by weight of disodium hydrogen phosphate, 0.12 parts by weight of sodium dihydrogen phosphate, 6 parts by weight of mannitol and 80 parts by weight of purified water were mixed, filtered, and sterilized at 120° C. for 30 min to obtain phase A, which was allowed to stand and cool to 38° C. for later use;

[0059] Take one-third of phase A and mix with 1 part by weight of carnosine, 0.3 part by weight of blue copper peptide, 0.001 part by weight of fibronectin, and 0.02 part by weight of soluble collagen, filter to obtain phase C, mix phase C and the remaining two-thirds of phase A to obtain a lyophilized powder liquid, and dispense the lyophilized powder liquid into lyophilized powder bottles, with a canning amount of 1.6 mL / piece;

[0060] The freeze-dried powder liquid is freeze-dried to obtain freeze-dried powder.

[0061] The filtration during the preparation of freeze-dried powder in Example 3 was performed using a 0.20 μm filter device.

[0062] In the freeze-dried powder preparation process of Example 3, the freeze-drying process comprises the following steps:

[0063] Pre-freezing treatment: the temperature of the pre-freezing treatment is -35°C and the time of the pre-freezing treatment is 2h;

[0064] Vacuum treatment, the setting value of vacuum treatment is 0.5mbar, and the vacuum treatment time is 1h;

[0065] Sublimation treatment, the sublimation treatment includes a first sublimation treatment, a second sublimation treatment and a third sublimation treatment in sequence, the temperature of the first sublimation treatment is -15°C, the time of the first sublimation treatment is 8 hours, the temperature of the second sublimation treatment is -3°C, the time of the second sublimation treatment is 4 hours, the temperature of the third sublimation treatment is 0°C, and the time of the third sublimation treatment is 0.5 hours;

[0066] The heating treatment includes a first heating treatment, a second heating treatment and a third heating treatment in sequence. The temperature of the first heating treatment is 4°C, the temperature of the second heating treatment is 13°C, the temperature of the third heating treatment is 28°C, and the time of the first heating treatment, the second heating treatment and the third heating treatment is 1 hour;

[0067] The insulation treatment temperature is 25°C and the insulation treatment time is 5h.

[0068] Example 4

[0069] A freeze-dried powder comprises the following raw materials in parts by weight: 0.1 part of sodium hyaluronate, 0.002 parts of oligopeptides, 1 part of carnosine, 0.001 parts of snake venom peptide, 0.001 parts of acetyl hexapeptide-8, 0.5 parts of serum albumin, 0.437 parts of disodium hydrogen phosphate, 0.12 parts of sodium dihydrogen phosphate, 5 parts of mannitol, 0.5 parts of HP skin wrinkle smoothing agent and 1 part of bosine.

[0070] The freeze-dried powder preparation method of Example 4 is as follows:

[0071] Mix 0.437 parts by weight of disodium hydrogen phosphate, 0.12 parts by weight of sodium dihydrogen phosphate, 6 parts by weight of mannitol, 0.5 parts by weight of HP skin wrinkle smoothing agent and 80 parts by weight of purified water, filter, sterilize at 120° C. for 30 min to obtain phase A, and cool phase A to 38° C. for later use;

[0072] Mix 0.1 parts by weight of sodium hyaluronate, 1 part by weight of bosine and 11 parts by weight of purified water, and filter to obtain phase B;

[0073] Take one third of phase A and mix it with 0.002 weight parts of oligopeptide, 1 weight part of carnosine, 0.001 weight part of snake venom peptide, 0.001 weight part of acetyl hexapeptide-8, and 0.5 weight part of serum albumin, filter it to obtain phase C, mix phase C, the remaining two thirds of phase A and phase B to obtain a lyophilized powder liquid, and pack the lyophilized powder liquid into lyophilized powder bottles, with a canning amount of 1.6 mL / tube;

[0074] The freeze-dried powder liquid is freeze-dried to obtain freeze-dried powder.

[0075] In the freeze-dried powder preparation process of Example 4, filtration was performed using a 0.20 μm filter device.

[0076] In the freeze-dried powder preparation process of Example 4, the freeze-drying process comprises the following steps:

[0077] Pre-freezing treatment: the temperature of the pre-freezing treatment is -35°C and the time of the pre-freezing treatment is 2h;

[0078] Vacuum treatment, the setting value of vacuum treatment is 0.5mbar, and the vacuum treatment time is 1h;

[0079] Sublimation treatment, the sublimation treatment includes a first sublimation treatment, a second sublimation treatment and a third sublimation treatment in sequence, the temperature of the first sublimation treatment is -15°C, the time of the first sublimation treatment is 8 hours, the temperature of the second sublimation treatment is -3°C, the time of the second sublimation treatment is 4 hours, the temperature of the third sublimation treatment is 0°C, and the time of the third sublimation treatment is 0.5 hours;

[0080] The heating treatment includes a first heating treatment, a second heating treatment and a third heating treatment in sequence. The temperature of the first heating treatment is 4°C, the temperature of the second heating treatment is 13°C, the temperature of the third heating treatment is 28°C, and the time of the first heating treatment, the second heating treatment and the third heating treatment is 1 hour;

[0081] The insulation treatment temperature is 25°C and the insulation treatment time is 5h.

[0082] Evaluation of the efficacy of the freeze-dried powder prepared in Example 1 on skin repairing effect:

[0083] 1. Purpose and Principle of Evaluation

[0084] At least 30 subjects with sensitive skin were recruited to use the test samples for 28 consecutive days. Subjective and semi-subjective evaluations (lactic acid stinging test) were performed, and facial image analyzer VISIA-CR was used to collect pictures of the red areas on the subjects' faces. The trans-epidermal water loss (TEWL) value of the skin was measured using the trans-epidermal water loss test probe Tewa meter TM Hex. The changes in various indicators before and after use were compared to determine the repair effect of the product.

[0085] 2 Subjects and related standards

[0086] 2.1 Subjects

[0087] A total of 33 subjects were included in this example (33 subjects were enrolled and 0 subjects dropped out), including 32 females and 1 male, aged 22 to 59 years old, with an average age of 40.88±11.05 years old, which met the subject inclusion criteria.

[0088] 2.2 Subject inclusion criteria

[0089] (1) Age range: 18 to 60 years old, both men and women;

[0090] (2) Being in good general health and free from major systemic diseases;

[0091] (3) Volunteers with sensitive skin and positive reaction to lactic acid sting test;

[0092] (4) Able to strictly follow the requirements and schedule of the research plan and sign the informed consent and confidentiality agreement;

[0093] 2.3 Subject Exclusion Criteria

[0094] (1) Women who are currently pregnant, breastfeeding, or planning to become pregnant in the next two months;

[0095] (2) Acute facial inflammation or other skin diseases, such as rosacea, eczema, lupus erythematosus, seborrheic dermatitis, psoriasis, severe epidermal exfoliation, etc.;

[0096] (3) Those with highly sensitive constitution or allergy to cosmetics;

[0097] (4) Those with obvious sunburn, scars, excessive hair growth on their faces or other conditions that may affect the test results;

[0098] (5) Any medications taken orally or topically in the past month that may affect skin condition or reaction, such as antihistamines, antibiotics, insulin, anti-inflammatory drugs, vitamin A, steroid drugs, aspirin, thyroid drugs, etc.;

[0099] (6) Those who have used hormone drugs and immunosuppressants in the past month;

[0100] (7) Those whose test sites are currently or have participated in other clinical trials in the past month;

[0101] (8) Have undergone facial cosmetic treatments such as chemical peel (fruit acid treatment), microdermabrasion, laser resurfacing, etc. within three months.

[0102] 2.4 Subject Exclusion Criteria

[0103] Serious adverse reactions occur during the trial and those who do not comply with the R&D plan, such as using other cosmetics or drugs during the test and requesting to withdraw for other reasons.

[0104] 3. Preparation before evaluation test

[0105] No products can be used on the test site on the day of the test. Before the test, the subjects need to clean their faces uniformly, dry them with dandruff-free tissues, and sit quietly in the human efficacy evaluation room (temperature: 20-22°C, humidity: 40-60%) for 30 minutes, and cannot drink water or beverages during this period. The subjects should remain relaxed, expose their faces, and avoid touching them.

[0106] Before the test, the subjects were given an explanation of the test and signed an informed consent form. The test area was the face.

[0107] 4How to use the product

[0108] After cleansing, pour the lysozyme into the freeze-dried powder, shake well until completely dissolved, and apply an appropriate amount evenly on the face until fully absorbed. Use once in the morning and evening every day, one pair every 4 days, and use it continuously for 28 days. During the trial, the subjects are prohibited from using repair preparations; the subjects are prohibited from dripping, injecting, orally or taking other forms of preparations that affect the repair test; the subjects are mainly indoor activities and avoid long-term exposure to outdoor light.

[0109] 5 Subjects’ subjective evaluation

[0110] During the follow-up visit 28 days after using the product (D28), the subjects evaluated their facial condition through a mirror under natural light conditions in the human efficacy laboratory and recorded the score. The seven-point scoring method was used to score the product in four aspects: skin feel, use effect, mildness, and use satisfaction. The judgment was then made and recorded once for each follow-up visit.

[0111] 6 Semi-subjective evaluation (lactic acid sting test)

[0112] On the day of enrollment and at each follow-up visit, 50 μL of 10% lactic acid solution was placed on filter paper (0.8 cm × 0.8 cm) at room temperature and placed on the nasolabial groove and any cheek. The subjects' sensitive reactions were rated at 4 levels at 0.5, 2.5, and 5 minutes, and then the three scores were added together. A score greater than 3 was considered a positive result. Those with a positive lactic acid sting test result can be judged as having sensitive skin and included in this test.

[0113] 0 means no stinging pain; 1 means mild stinging pain; 2 means moderate stinging pain; 3 means severe stinging pain.

[0114] 7 Objective quantitative evaluation

[0115] 30 minutes after facial cleansing, facial image analyzer VISIA-CR was used to take facial photos, and the transepidermal water loss test probe Tewameter TM Hex was used to test the skin TEWL value at the test site. The measured value was used as the baseline value (D0), and then the product was distributed and the usage instructions were given. The subjects needed to return for a visit 28 days after using the product (D28) and undergo the same indicator test.

[0116] The test of the same subject should be completed by the same instrument and the same tester. The test site should be kept consistent between the two test time points.

[0117] 8 Statistical analysis methods

[0118] The measured values ​​of each test area are statistically analyzed, including quantity, mean, standard deviation, standard error, minimum value, median, maximum value, etc. If the test data is normally distributed, the paired t-test method is used for statistical analysis; if the test data is non-normally distributed, the rank sum test method is used for statistical analysis.

[0119] The statistical methods all adopted two-tailed test, and the test level was α=0.05.

[0120] SPSS 25.0 software was used for statistical analysis. When the results showed significant differences, *p<0.05 and **p<0.01 were used to mark them. *p<0.05 indicated a significant difference, and **p<0.01 indicated an extremely significant difference. If there were significant differences in the values ​​of the indicators in the test area, it means that the product has a repairing effect.

[0121] 9 Evaluation test results

[0122] 9.1 Safety and adverse reaction evaluation

[0123] During the trial, professional dermatologists asked the subjects whether they experienced dryness, oiliness, scaling, redness, stinging and other symptoms while using the tested products, and observed and recorded whether there were rashes, redness, swelling and scaling on the face. Then the scores were scored according to the scoring criteria in Table 1, and the scores were recorded in the event report form, and the correlation was evaluated according to the criteria listed in Table 2. During the trial, no subjects experienced adverse reactions.

[0124] Table 1 Scoring criteria for adverse reactions

[0125]

[0126] Table 2 Evaluation of the tested products

[0127]

[0128] 9.2 Subjective evaluation

[0129] The subjective evaluation results of the subjects are shown in Figure 1 .

[0130] 9.3 Semi-subjective evaluation (lactic acid sting test)

[0131] At room temperature, 50 μL of 10% lactic acid solution was placed on filter paper (0.8 cm × 0.8 cm) and placed on the nasolabial groove and any side of the cheek. The subjects' sensitivity was rated at 4 levels at 0.5, 2.5 and 5 minutes, and then the 3 scores were added together. The smaller the score value, the lower the degree of lactic acid stimulation.

[0132] Table 3 Lactic acid sting test results (n=33)

[0133]

[0134]

[0135] The results are shown in Table 3. Compared with the baseline value before use (D0), the mean value of the lactic acid sting test result showed a downward trend after using the product, and decreased by 29.29% after 28 days of use (D28), which was significantly different from the baseline value (p<0.01).

[0136] 9.4 Analysis of Transepidermal Water Loss (TEWL) Values

[0137] The larger the TEWL value measured by the transepidermal water loss test probe Tewameter TM Hex, the more transepidermal water loss per unit time and per unit cross-sectional area, and vice versa. Therefore, the trend of decreasing TEWL value represents the recovery process of transepidermal water loss of barrier-damaged skin. By comparing the changes in the average TEWL value of the skin before and after use, the effect of the product in reducing skin water loss can be reflected.

[0138] The more the average TEWL value at different follow-up time points decreases compared with the baseline value before use (D0), the better the repair effect of the product.

[0139] TEWL value change rate = (TEWL value after using the product - TEWL value before using the product) / TEWL value before using the product × 100%.

[0140] Table 4 Descriptive statistics of the mean values ​​of skin TEWL (g / (hm 2 ), n=33)

[0141] Group Number of people average value Standard Deviation Standard error Minimum Maximum median D0 33 25.75 4.59 0.80 19.44 36.69 24.30 D28 33 22.53 4.98 0.87 13.50 34.28 21.94

[0142] Table 5 Analysis of the difference in the average values ​​of skin TEWL (g / (hm 2 ), x±s, n=33)

[0143] Group Mean ± SD p-value (vs.D0) D0 25.75±4.59 / D28 22.53±4.98 <0.01

[0144] Compared with the baseline value before use (D0), the average skin TEWL value showed a downward trend after using the product, and decreased by 12.47% after 28 days of use (D28), which was significantly different from the baseline value (p<0.01). The average skin TEWL value after using the product was lower than the baseline value.

[0145] 10 Evaluation Conclusion

[0146] (1) No adverse reactions occurred in this trial, and the trial product has a high degree of safety.

[0147] (2) The results of the semi-subjective evaluation (lactic acid sting test) showed that after the subjects used the product, the mean value of the lactic acid sting test showed a downward trend at the follow-up time point D28, which was significantly different from the baseline value.

[0148] (3) Subjective evaluations by the subjects showed that after 28 days of use (D28), the mean scores were all above 5 points, and statistically more than 50% of the subjects gave positive comments on the product's skin feel, soothing effect, mildness, and satisfaction. At D28, more than 90% of the subjects agreed with "skin does not feel tight after use", "the product is mild and non-irritating", "suitable for sensitive skin", "soothes burning sensation on the skin", "relieves stinging and itching", "improves skin redness", and "improves skin tolerance". And 100% of the subjects gave positive comments on product satisfaction.

[0149] (4) Analysis of skin TEWL values ​​showed that after the subjects used the product, the TEWL values ​​showed a downward trend at the follow-up time point D28, which was significantly different from the baseline value.

[0150] In summary, after using the freeze-dried powder of Example 1 for 28 days, the stinging reaction caused by lactic acid on subjects with sensitive skin can be improved, the moisture loss rate of facial skin can be reduced, and the repair effect can be achieved. In the subjective evaluation, more than 90% of the subjects gave positive comments on the product's skin feel, soothing effect, mildness and satisfaction, indicating that the product has a repairing effect and is suitable for sensitive skin.

[0151] Evaluation of the efficacy of the lyophilized powder prepared in Example 2 on its firming and anti-wrinkle effects:

[0152] 1 Evaluation Principle

[0153] Elastin plays a key role in the youthfulness of the skin, and the lack of elastin is the main cause of skin aging. Inhibiting the activity of elastase can effectively slow down the decomposition of elastin and maintain the elasticity of the skin. Elastase can catalyze the hydrolysis of hydroxyl groups contained in the polypeptide bonds of various amino acids. Therefore, when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, it will produce colored substances, and its absorbance can be measured at 410nm in a spectrophotometer. According to the change in absorbance, the inhibitory effect of the test substance on the activity of elastase is calculated, and whether the test substance has firming and anti-wrinkle effects can be evaluated.

[0154] 2. Evaluation test equipment and reagents

[0155] Instruments and equipment: 1 / 10,000 precision balance, microplate reader.

[0156] Reagents: elastase, colorimetric substrate AAAPAN (N-succinyl-alanine-alanine-alanine-p-nitroanilide), positive control EGCG (epigallocatechin gallate), buffer Tris-HCl (pH=8.0).

[0157] 3 Experiments

[0158] 3.1 Test operation

[0159] Elastase and chromogenic substrate AAAPAN were prepared into working solutions using buffer Tris-HCl and prepared immediately before use.

[0160] The positive control EGCG was prepared with buffer and diluted to a series of concentrations in half times to verify the test system.

[0161] The test substance was treated and diluted with buffer to the required test concentration, or not diluted if no requirement was given.

[0162] The sample addition grouping is shown in Table 6. A 96-well microplate is used for the test, and sample group T, sample background group T0, enzyme reaction group C, and solvent background group C0 are set up. The positive control group is treated the same as the sample group. Three parallels are set up for each group.

[0163] Table 6 Sample liquid addition grouping table

[0164]

[0165]

[0166] Immediately use the microplate reader to detect, set the parameters: slow oscillation for 10 seconds, OD410nm absorbance measurement. Export the file after the instrument completes the reading.

[0167] 3.2 Result calculation

[0168] Calculate the inhibition rate of elastase activity:

[0169]

[0170] Where:

[0171] T—absorbance value of sample group, i.e., the absorbance value of the solution after the sample affects the reaction between elastase and substrate AAAPAN;

[0172] T0—absorbance value of sample background group;

[0173] C—absorbance value of enzyme reaction group, i.e., absorbance value of the reaction between elastase and substrate AAAPAN when no sample is added;

[0174] C0—Solvent background group absorbance value.

[0175] 3.3 Results

[0176] The experimental data are expressed as mean±SD, and statistical methods such as t-test can be used to analyze the data.

[0177] 4 Test results

[0178] The test results are shown in Table 7.

[0179] Table 7 Elastase activity inhibition rate results

[0180]

[0181] Compared with the blank group: *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

[0182] 5 Evaluation conclusion

[0183] As shown in Table 7, the 100% (V / V) test group had an inhibitory effect on the decomposition of AAAPAN catalyzed by elastase, with an inhibition rate of 22.17±1.68%, and the data had a significant difference (p=0.0030).

[0184] The test results can be used as one of the evidences to support that the freeze-dried powder of Example 2 achieves firming and anti-wrinkle effects by inhibiting the activity of elastase.

[0185] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A freeze-dried powder, characterized in that: The invention comprises the following raw materials in parts by weight: 0-10 parts of polysaccharide, 0-3 parts of hyaluronic acid, 0.0001-1.5 parts of polypeptide active substance, 0.01-1.5 parts of protein, 0.1-0.7 parts of buffer and 2-10 parts of excipient.

2. The freeze-dried powder according to claim 1, characterized in that The polysaccharide comprises at least one of Tremella heteropolysaccharide, soluble proteoglycan, trehalose and Tremella polysaccharide.

3. The freeze-dried powder according to claim 1, characterized in that The hyaluronic acid includes at least one of hydrolyzed hyaluronic acid and sodium hyaluronate.

4. The freeze-dried powder according to claim 1, characterized in that The polypeptide active substance includes at least one of oligopeptide, oligopeptide-3, oligopeptide-2, oligopeptide-4, snake venom peptide, blue copper peptide, acetyl hexapeptide-8, carnosine, pentapeptide-3 and tripeptide-1.

5. The freeze-dried powder according to claim 1, characterized in that The protein includes at least one of hydrolyzed collagen, type I recombinant collagen, type II recombinant collagen, type III recombinant collagen, fibronectin and serum albumin.

6. The freeze-dried powder according to claim 1, characterized in that The buffer includes at least one of disodium hydrogen phosphate and sodium dihydrogen phosphate.

7. The freeze-dried powder according to claim 1, characterized in that The excipients include mannitol.

8. A method for preparing a freeze-dried powder according to any one of claims 1 to 7, characterized in that: The following steps are involved: The polysaccharide, the buffer, the excipient and water are mixed, filtered and sterilized to obtain phase A; Mixing the hyaluronic acid and water, filtering, and obtaining phase B; Taking part of the phase A, mixing it with the polypeptide active substance and the protein, filtering it to obtain phase C, and mixing the phase C, the remaining phase A and the phase B to obtain a lyophilized powder liquid; The freeze-dried powder liquid is freeze-dried to obtain the freeze-dried powder.

9. The preparation method according to claim 8, characterized in that: Filtration is performed using a 0.20-0.50 μm filter device.

10. The preparation method according to claim 8, characterized in that: The freeze-drying process comprises the following steps: Pre-freezing treatment, wherein the temperature of the pre-freezing treatment is -35 to 55°C and the time of the pre-freezing treatment is 2 to 3 hours; Vacuum treatment; Sublimation treatment, the sublimation treatment includes a first sublimation treatment, a second sublimation treatment and a third sublimation treatment in sequence, the temperature of the first sublimation treatment is -15 to 25°C, the time of the first sublimation treatment is 8 to 10 hours, the temperature of the second sublimation treatment is -3 to 8°C, the time of the second sublimation treatment is 4 to 6 hours, the temperature of the third sublimation treatment is 0°C, and the time of the third sublimation treatment is 0.5 to 1.5 hours; Temperature treatment, wherein the temperature treatment includes a first temperature treatment, a second temperature treatment and a third temperature treatment in sequence, the temperature of the first temperature treatment is 4 to 6°C, the temperature of the second temperature treatment is 13 to 17°C, the temperature of the third temperature treatment is 28 to 33°C, and the time of the first temperature treatment, the second temperature treatment and the third temperature treatment are all 1 to 2.5 hours; The heat preservation treatment is performed at a temperature of 25 to 45° C. and a time of 5 to 9 hours.

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