CN117297996A - Hirudin freeze-dried mask and preparation method thereof - Google Patents

Abstract

The invention discloses a hirudin freeze-dried facial mask and a preparation method thereof. The hirudin facial mask prepared by using the hirudin freeze-dried facial mask preparation method provided by the invention has been experimentally confirmed to have clear anti-aging and whitening effects; it overcomes the problem of leeches. It has the disadvantages of being unstable in water, easily deactivated, deteriorating, and having a fishy smell at room temperature; after purification and removal of the fishy smell, it is prepared into a freeze-dried facial mask for storage and use, which improves stability and solves the problem of long-term storage and use of active ingredients added to cosmetics. The problem of odor; the freeze-dried facial mask provided by the present invention has stable and effective activity, avoids the addition of unnecessary preservatives, etc., and reduces the burden on the skin.

Description

A hirudin freeze-dried facial mask and preparation method thereof

Technical Field

The invention belongs to the technical field of skin care products and preparation thereof, and specifically relates to a hirudin freeze-dried facial mask and a preparation method thereof.

Background Art

Facial mask refers to a facial skin cosmetic that is applied to the surface of human skin and peeled off, scrubbed or retained after a period of time. It plays a role in concentrated cleaning, care and nutrition. It is deeply loved by adults, especially female consumers. With the maturity of consumer psychology, people not only require facial masks to achieve the basic functions of cleaning, care and nutrition, but also further pursue the development of advanced functions such as whitening and anti-aging. At present, there are many types of facial masks, including sticker masks, tear-off masks, gel masks, freeze-dried masks, etc. Among them, freeze-dried masks are also called freeze-dried powder masks and freeze-dried solid liquid masks. It refers to the fusion of mask liquid, thickeners, moisturizers and other ingredients with the fiber of the membrane cloth, using vacuum freeze drying to sublimate the water and form a solid dry film. After cleansing, take out the freeze-dried mask directly and apply it on the face, leave it for about 20 minutes, gently massage the remaining essence, and apply it on the hands, feet or other parts of the body. After the essence on the face is massaged and absorbed, wash it off with clean water. At the same time, due to the good physical properties of the freeze-dried mask, it can be a good drug carrier. Adding different drugs into it can achieve multiple effects of hydration and beauty.

Leeches are highly specialized annelids. They were first recorded in Shennong's Herbal Classics. They are commonly known as leeches. They are traditional Chinese medicines with salty and bitter tastes and are neutral in nature. The Pharmacopoeia of the People's Republic of my country states that they have the effects of breaking blood, removing blood stasis, and promoting menstruation. They are mainly used to treat tumors, lumps, blood stasis, amenorrhea, and traumatic injuries. In 1884, Haycraft first discovered a biologically active substance, hirudin, in the salivary glands of European medicinal leeches. In 1927, Shionoya first studied it as an effective antithrombotic drug. In 1955, Markwardt pointed out that it is a protein substance and named it hirudin. Natural hirudin is an anticoagulant protein extracted from medical leeches. It is a single-chain polypeptide composed of 65 or 66 amino acid residues with a relative molecular mass of 7000 Dalton.

Hirudin itself has a strong fishy smell, so it is difficult to apply it to skin care products. In the prior art, hirudin is mainly used in ordinary patch masks and smear masks, both of which belong to liquid environments and require the addition of a certain amount of preservatives. Some people's skin is irritated and allergic to preservatives. Hirudin is a polypeptide that is also easily inactivated and deteriorated in a liquid environment.

Summary of the invention

In order to solve the above technical problems, the present invention provides a freeze-dried facial mask using hirudin as an active ingredient, which has been experimentally confirmed to have clear anti-aging and whitening effects; it overcomes the shortcomings of hirudin being unstable in water, easily inactivated, deteriorated, and having a fishy smell at room temperature; after purification and removal of the fishy smell, it is prepared into a freeze-dried facial mask for storage and use, which improves stability and solves the problem that active ingredients added to cosmetics cannot be preserved for a long time and have a bad smell; the freeze-dried facial mask provided by the present invention is active, stable and effective, avoids the addition of unnecessary preservatives, etc., and reduces the burden on the skin.

In order to achieve the above technical purpose, the present invention is implemented by the following technical scheme: a hirudin freeze-dried facial mask, including a hirudin anti-aging freeze-dried facial mask and a hirudin whitening freeze-dried facial mask;

The hirudin anti-aging freeze-dried mask is composed of active ingredients and auxiliary ingredients. The active ingredients include: 0.2-8 parts of hirudin extract; the auxiliary ingredients include: 1-99 parts of water, 1-50 parts of hydroxyethyl urea, 1-30 parts of sodium hyaluronate, 0.1-20 parts of jojoba seed oil, 0.25-10 parts of glycerol, 0.25-10 parts of trehalose, 0.2-10 parts of betaine, 0.1-8 parts of hydroxyethyl cellulose, 0.05-7 parts of oligofructose, 0.05-7 parts of collagen, 0.05-5 parts of squalane, 0.01-5 parts of polyglycerol-2 oleate, and 0.01-2 parts of Tremella fruiting body extract;

The hirudin whitening freeze-dried mask is composed of active ingredients and auxiliary ingredients. The active ingredients include: hirudin extract, collagen, oligopeptide-10, oligopeptide-3, and allantoin; the auxiliary ingredients include: mannitol, trehalose, sodium hyaluronate with a molecular weight of 8,000 Daltons, sodium hyaluronate with a molecular weight of 1.3 million Daltons, disodium hydrogen phosphate, sodium dihydrogen phosphate, glycerin, and solvents.

Preferably, the hirudin extract is composed of 3 parts, water 50 parts, hydroxyethyl urea 20 parts, sodium hyaluronate 10 parts, jojoba seed oil 5 parts, glycerol 3 parts, trehalose 3 parts, betaine 3 parts, hydroxyethyl cellulose 1 part, collagen 0.8 parts, squalane 0.7 parts, polyglycerol-2 oleate 0.3 parts, and Tremella fuciformis fruiting body extract 0.2 parts;

Preferably, the hirudin extract is 1.2 parts, water is 70 parts, hydroxyethyl urea is 9 parts, sodium hyaluronate is 6 parts, jojoba seed oil is 5 parts, glycerol is 2 parts, trehalose is 2 parts, betaine is 1.2 parts, oligofructose is 1.2 parts, hydroxyethyl cellulose is 0.7 parts, oligofructose is 0.6 parts, collagen is 0.54 parts, squalane is 0.52 parts, polyglycerol-2 oleate is 0.03 parts, and Tremella fruiting body extract is 0.01 parts;

Preferably, the hirudin extract 1%, collagen 1.5%, oligopeptide-10.5%, oligopeptide-30.5%, allantoin 0.5%; mannitol 2.5%, trehalose 2.5%, sodium hyaluronate with a molecular weight of 8000 Daltons 0.25%, sodium hyaluronate with a molecular weight of 1.3 million Daltons 0.25%, disodium hydrogen phosphate 0.15%, sodium dihydrogen phosphate 0.12%, glycerol 2%, solvent;

Preferably, the hirudin extract 1.5%, collagen 1.8%, oligopeptide-10.6%, oligopeptide-30.8%, allantoin 0.6%; mannitol 3%, trehalose 2.5%, sodium hyaluronate with a molecular weight of 8000 Daltons 0.28%, sodium hyaluronate with a molecular weight of 1.3 million Daltons 0.28%, disodium hydrogen phosphate 0.25%, sodium dihydrogen phosphate 0.14%, glycerol 3%, solvent;

Preferably, the hirudin extract 2%, collagen 2%, oligopeptide-11%, oligopeptide-31.5%, allantoin 0.8%; mannitol 3%, trehalose 3.5%, sodium hyaluronate with a molecular weight of 8000 Daltons 0.35%, sodium hyaluronate with a molecular weight of 1.3 million Daltons 0.29%, disodium hydrogen phosphate 0.25%, sodium dihydrogen phosphate 0.15%, glycerol 4%, solvent;

Preferably, the solvent is deionized water or purified water.

Another object of the present invention is to provide a method for preparing a hirudin freeze-dried facial mask, comprising the following steps:

S1: dissolving the active ingredient in distilled water and filtering through a 0.22 μm pore size filter membrane to obtain solution 1;

S2: dissolving the excipients in deionized water to obtain solution 2;

S3: Mixing solution 1 and solution 2 to obtain a facial mask essence;

S4: Disinfect the mask cloth, fold it and immerse it in the mask essence; let it stand for 30 to 35 minutes until the mask cloth fully absorbs the mask essence;

S5: Push the processed semi-finished product tray into the freeze dryer, quickly cool it to -20 to -80°C, and keep it for 2 to 6 hours;

S6: After the base fabric and the material body are completely crystallized, vacuum is drawn for sublimation drying; the sublimation drying time is 20 to 28 hours, and after drying, the air is released and the material is taken out of the box;

S7: quickly packing the freeze-dried product into a facial mask packaging bag, sterilizing the inside of the facial mask packaging bag with cobalt radiation, evacuating the facial mask packaging bag under a nitrogen environment, and sealing the facial mask packaging bag to obtain the hirudin freeze-dried facial mask product.

The beneficial effects of the present invention are:

Compared with the prior art, the hirudin freeze-dried facial mask provided by the present invention overcomes the problems that effective ingredients such as peptides are easily inactivated in a liquid environment; preservatives must be added in liquid form, which may cause irritation and allergies to the skin; and the problem of product ingredients and instability in liquid form; the active raw materials of the product are stable and effective, avoiding the addition of unnecessary preservatives, etc., and are more friendly to the skin; and it has been verified by experiments that it has clear anti-aging and whitening effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the comparison of anticoagulant activity results;

FIG2 is a diagram showing the results of the fishy smell sensory scoring;

FIG3 is a graph showing the comparison of DPPH free radical elimination rates of hirudin and VC;

FIG4 is a graph showing the comparison of the elimination rates of hydroxyl radicals of hirudin and VC;

FIG5 is the effect of hirudin and arbutin on tyrosinase activity in B16 cells;

FIG6 is the effect of hirudin and arbutin on melanin synthesis in B16 cells;

FIG7 is a diagram showing the result of establishing a chloasma model;

FIG8 is a diagram showing the results of HE staining of the normal group and the model group to verify the successful modeling;

FIG9 is a schematic diagram showing the changes in back skin lesions after progesterone administration and ultraviolet irradiation for 30 days;

FIG10 is a schematic diagram of the gray value ratio results after drug administration;

FIG. 11 is a schematic diagram of the MDA content comparison results.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

In this embodiment, the raw materials are weighed in sequence, 6.437 kg of water, 0.8 kg of hydroxyethyl urea, 0.7 kg of sodium hyaluronate, 0.6 kg of jojoba seed oil, 0.5 kg of glycerol, 0.3 kg of trehalose, 0.2 kg of betaine, 0.2 kg of hirudin, 0.1 kg of hydroxyethyl cellulose, 0.05 kg of oligofructose, 0.05 kg of collagen, 0.032 kg of squalane, 0.021 kg of polyglycerol-2 oleate, and 0.01 kg of Tremella fruiting body extract group. The freeze-dried mask product is prepared according to the following steps:

The raw materials weighed above are used to prepare a freeze-dried facial mask according to the following steps:

S1: 4 kg of water, hydroxyethyl urea, sodium hyaluronate, jojoba seed oil, glycerin, trehalose, betaine, hydroxyethyl cellulose, oligofructose, collagen, squalane, polyglycerol-2 oleate, Tremella fuciformis fruiting body extract and other raw materials are prepared into an aqueous solution according to a proportion to obtain solution 1;

S2: dissolving hirudin in the remaining water and filtering with a filter membrane with a pore size of 0.22 μm to obtain solution 2;

S3: Evenly mix solution 1 and solution 2 to obtain a facial mask essence;

S4: Disinfect the mask cloth, fold it according to a fixed folding method and soak it in the mask essence;

S5: Let it stand for 30 minutes until the mask base fabric fully absorbs the material;

S6: Push the processed semi-finished product into the freeze dryer, quickly cool it to -20°C, and keep it for 2 hours;

S7: After the base fabric and the material inside the bottle are completely crystallized, vacuum 23pa is started for sublimation drying;

S8: The sublimation drying time is 21 hours, and the air is released and the box is opened after drying;

S9: quickly packing the freeze-dried product into a facial mask packaging bag, disinfecting and sterilizing the facial mask packaging bag, evacuating the facial mask packaging bag under a nitrogen environment, and sealing the facial mask packaging bag to obtain the hirudin freeze-dried facial mask product.

Example 2

In this embodiment, the raw materials are weighed in sequence, 6 kg of water, 1 kg of hydroxyethyl urea, 0.8 kg of sodium hyaluronate, 0.5 kg of jojoba seed oil, 0.4 kg of glycerol, 0.3 kg of trehalose, 0.3 kg of betaine, 0.25 kg of hirudin, 0.2 kg of hydroxyethyl cellulose, 0.1 kg of oligofructose, 0.1 kg of collagen, 0.03 kg of squalane, 0.01 kg of polyglycerol-2 oleate, and 0.01 kg of Tremella fruiting body extract group. The freeze-dried mask product is prepared according to the following steps:

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -30°C and maintained for 2.5 hours; a vacuum of 30 Pa was applied for sublimation drying for 25 hours.

Example 3

In this embodiment, the raw materials are weighed in sequence, including 7 kg of water, 0.6 kg of hydroxyethyl urea, 0.5 kg of sodium hyaluronate, 0.45 kg of jojoba seed oil, 0.35 kg of glycerol, 0.32 kg of trehalose, 0.27 kg of betaine, 0.18 kg of hirudin, 0.1 kg of hydroxyethyl cellulose, 0.08 kg of oligofructose, 0.07 kg of collagen, 0.05 kg of squalane, 0.015 kg of polyglycerol-2 oleate, and 0.015 kg of Tremella fruiting body extract group. The freeze-dried mask product is prepared according to the following steps:

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -40°C and maintained for 1.9 hours; a vacuum of 45 Pa was applied for sublimation drying for 18 hours.

Example 4

In this embodiment, the raw materials are weighed in sequence, including 7.017 kg of water, 0.68 kg of hydroxyethyl urea, 0.55 kg of sodium hyaluronate, 0.47 kg of jojoba seed oil, 0.32 kg of glycerol, 0.27 kg of trehalose, 0.22 kg of betaine, 0.19 kg of hirudin, 0.08 kg of hydroxyethyl cellulose, 0.07 kg of oligofructose, 0.06 kg of collagen, 0.05 kg of squalane, 0.012 kg of polyglycerol-2 oleate, and 0.011 kg of Tremella fruiting body extract group. The freeze-dried mask product is prepared according to the following steps:

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -45°C and maintained for 2.3 hours; a vacuum of 48 Pa was applied for sublimation drying for 23 hours.

Example 5

In this embodiment, the raw materials are weighed in sequence, including 8.011 kg of water, 0.5 kg of hydroxyethyl urea, 0.32 kg of sodium hyaluronate, 0.28 kg of jojoba seed oil, 0.21 kg of glycerol, 0.2 kg of trehalose, 0.145 kg of betaine, 0.14 kg of hirudin, 0.06 kg of hydroxyethyl cellulose, 0.05 kg of oligofructose, 0.041 kg of collagen, 0.032 kg of squalane, 0.006 kg of polyglycerol-2 oleate, and 0.005 kg of Tremella fruiting body extract group. The freeze-dried mask product is prepared according to the following steps:

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -48°C and maintained for 1.8 hours; a vacuum of 50 Pa was applied for sublimation drying for 30 hours.

Example 6

In this embodiment, the functional raw materials, collagen, oligopeptide-1, oligopeptide-3, and hirudin are prepared in sequence. The auxiliary materials include, according to the final concentration, mannitol, trehalose, sodium hyaluronate with a molecular weight of 8,000 Daltons, sodium hyaluronate with a molecular weight of 1.3 million Daltons, disodium hydrogen phosphate, sodium dihydrogen phosphate, and glycerol. The solvent is deionized water.

Follow these steps to prepare the freeze-dried mask product:

Prepare the freeze-dried mask using the raw materials prepared above according to the following steps:

S1: Dissolve collagen, oligopeptide-1, oligopeptide-3 and hirudin in water according to the proportion to obtain solution A;

S2: Filter solution A through a filter membrane with a pore size of 0.22 μm to obtain solution 1;

S3: mixing part of the deionized water with mannitol, trehalose, sodium hyaluronate with a molecular weight of 8,000 Daltons, sodium hyaluronate with a molecular weight of 1.3 million Daltons, disodium hydrogen phosphate, and sodium dihydrogen phosphate to obtain a facial mask essence;

S4: Disinfect the mask cloth, fold it according to a fixed folding method and soak it in the mask essence;

S5: Let it stand for 30 minutes until the mask base fabric fully absorbs the material;

S6: Push the processed semi-finished product into the freeze dryer, quickly cool it to -20°C, and keep it for 2 hours;

S7: After the base fabric and the material inside the bottle are completely crystallized, vacuum 23pa is started for sublimation drying;

S8: The sublimation drying time is 21 hours, and the air is released and the box is opened after drying;

S9: quickly packing the freeze-dried product into a facial mask packaging bag, disinfecting and sterilizing the facial mask packaging bag, evacuating the facial mask packaging bag under a nitrogen environment, and sealing the facial mask packaging bag to obtain the hirudin freeze-dried facial mask product.

Example 7

In this embodiment, the functional raw materials are prepared in sequence, collagen 1.5%, oligopeptide-10.5%, oligopeptide-30.6%, hirudin 1.5%, and the auxiliary materials include, according to the final concentration composition: mannitol 2.5%, trehalose 2.5%, sodium hyaluronate with a molecular weight of 8000 Daltons 0.25%, sodium hyaluronate with a molecular weight of 1.3 million Daltons 15%, disodium hydrogen phosphate 0.14%, sodium dihydrogen phosphate 0.12%, glycerol 2%, and part of deionized water.

Follow these steps to prepare the freeze-dried mask product:

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -30°C and maintained for 3 hours; a vacuum of 30 Pa was applied for sublimation drying for 25 hours.

Example 8

In this embodiment, the functional raw materials are prepared in sequence, collagen 2.5%, oligopeptide-11.5%, oligopeptide-30.8%, hirudin 1.8%, and the auxiliary materials include, according to the final concentration composition: mannitol 2.8%, trehalose 2.5%, sodium hyaluronate with a molecular weight of 8000 Daltons 0.45%, sodium hyaluronate with a molecular weight of 1.3 million Daltons 0.15%, disodium hydrogen phosphate 0.18%, sodium dihydrogen phosphate 0.15%, glycerol 3%, and part of deionized water.

Follow these steps to prepare the freeze-dried mask product:

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -40°C and maintained for 2 hours; a vacuum of 45 Pa was applied for sublimation drying for 20 hours.

The freeze-dried facial mask was prepared according to the method of Example 1, except that the freeze dryer was rapidly cooled to -48°C and maintained for 1.8 hours; a vacuum of 50 Pa was applied for sublimation drying for 30 hours.

Example 9

Comparative Example 1

The difference between the facial mask prepared in Example 1 and Example 6 is that the comparative example 1 is not freeze-dried, and the contents of the remaining components and the preparation method are the same as those in Example 1 and Example 6. Hirudin is unstable in water, and after 7 days, it has a spoiled smell and its activity is reduced.

Comparative Example 2

The difference between the facial mask prepared in Example 1 and Example 6 is that the hirudin solution is not filtered, and the contents and preparation methods of the other components of Comparative Example 2 refer to those of Example 1 and Example 6. Failure to filter and sterilize cannot ensure compliance with the bacterial content of the product.

Comparative Example 3

The difference between the facial mask prepared in the above-mentioned embodiment 1 and embodiment 6 is that the solution 1 is sterilized at high temperature, and the contents and preparation methods of the other components are the same as those in embodiment 1 and embodiment 6. Hirudin is inactivated after high temperature sterilization.

Example 10

Hirudin deodorization process

Hirudin was extracted with a material-liquid ratio of 20:1g/ml, ultrasonic for 30min, extracted three times, and the extracts were combined. The combined supernatant was deodorized and freeze-dried before the sample hirudin was determined. First, titrate with a thrombin solution of 40NIH/mL, and titrate with a thrombin solution of 20NIH/mL near the end point. The volume of thrombin added was 1-5μL, and the titration volume of 1μL was used near the end point. The volume of thrombin solution added each time was 5μL, and the thrombin was stirred once with a syringe needle every time the thrombin was added, and the titration was completed every 1min, and the time limit was completed within 10min. Calculate the activity; sample solution activity = ∑ thrombin concentration × drop volume, and the sample hirudin content was converted from this. As shown in Figure 1, 1 is the original sample, 2 is the anticoagulant activity after water extraction, and 3-8 are the anticoagulant activities after treatment with different deodorization methods. Compared with 2, 3 has the best anticoagulant effect, and 2 and 3 are significantly different from 1 (P < 0.05).

Finally, a panel of 10 assessors (22-26 years old, 5 males and 5 females) conducted a sensory evaluation of this experiment. The fishy smell evaluation in this experiment adopted a 10-point system, with distilled water without fishy smell being scored as 0 points and untreated hirudin being scored as 10 points. During the evaluation, the sample was placed in a tube, and the assessors conducted a sensory evaluation according to the above scoring rules, and the average score was taken as the final score. Score 0-2: basically no fishy smell; 2-4: slightly fishy smell; 4-6: fishy smell; 6-8: obvious fishy smell; 8-10: strong fishy smell. The best deodorization process was screened based on antithrombin activity and sensory evaluation. The antithrombin activity of hirudin treated by this method was 511.1ATU/g, and the sensory evaluation score was 3 points. Conclusion: Purification method 3 has the best deodorization effect on hirudin, while preserving its antithrombin activity to the greatest extent. As shown in Figure 2, the original sample was treated with water extraction. Among the different deodorization effects of 2-8, the deodorization effect of 2 was the best compared with the original sample. Based on the anticoagulant effect of 2, 2 was determined to be the optimal deodorization and purification method.

Embodiment 11

Determination of antioxidant activity in vitro

Determination of DPPH free radical scavenging ability

Prepare mask samples with mass concentrations of 5, 2.5, 1.25, 0.625, and 0.3125 mg/mL with distilled water. Take 0.7 mL of each sample with different mass concentrations, add 1.3 mL of 0.3 mmol/L DPPH ethanol solution, and measure the absorbance of the mixture at a wavelength of 517 nm after the reaction is complete in the dark, with VC as the control; as shown in Figure 3;

Where: A0 is the absorbance value of the blank control, A1 is the absorbance value of the sample solution, and A2 is the absorbance value of anhydrous ethanol and the sample solution;

Determination of hydroxyl radical scavenging ability

Prepare mask samples with mass concentrations of 5, 2.5, 1.25, 0.625, and 0.3125 mg/mL with distilled water. Add 0.2 mL of 6 mmol/L ferrous sulfate solution and salicylic acid-ethanol solution to the reaction system in sequence, then add 0.2 mL of sample solutions of different mass concentrations and 0.2 mL of 6 mmol/L hydrogen peroxide solution, react at 37°C for 1 hour, and measure the absorbance of the mixture at a wavelength of 510 nm after the reaction is complete. As shown in Figure 4;

Wherein: A0 is the absorbance value of blank control, A1 is the absorbance value of sample, and A2 is the background absorption of sample without adding salicylic acid-ethanol solution.

Example 12

1) Determination of tyrosinase activity in B16 cells

96-well plate culture, add B16 single cell suspension (7-8) × 10 ³ cells/well, wait for 24 hours to adhere to the wall, add culture medium containing different mass concentration samples, and set up 3 replicates for each mass concentration. The control group used RPMI-1640 complete culture medium instead of sample solution. After incubation at 37°C, 5% CO ₂ for 72 hours, the supernatant was discarded. Wash twice with PBS with pH = 7.4, add 50μL of 1% octylphenol polyoxyethylene ether (Triton X-100) solution to each well, quickly freeze at -80°C for 30 minutes, then thaw at room temperature to completely rupture the cells, preheat at 37°C for 5 minutes, add 10μL of 1% L-DOPA solution, react at 37°C for 2 hours, and measure its absorbance at 490nm on an enzyme reader. Calculate the tyrosinase activity inhibition rate according to the following formula: inhibition rate = 1-(average absorbance of sample group/average absorbance of control group) × 100%.

2) Determination of melanin content in B16 cells

B16 cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 5×10 ⁴ cells/mL, 2 mL per well, and cultured for 24 hours. The culture medium of samples with different mass concentrations was replaced, and the control group was added with the same volume of fresh complete culture medium. Three parallel wells were set up for each group. After culturing for 72 hours at 37°C, 5% CO ₂ , and saturated humidity, the supernatant was discarded. Each well was washed once with PBS, and 0.5 mL of 5g/L trypsin digestion solution was added to digest in a 37°C incubator for 2-4 minutes. Observe under a microscope to ensure that all cells were digested. Add 1 mL of RPMI-1640 complete culture medium to terminate digestion, gently blow the cells to collect them in a 1.5 mL centrifuge tube, centrifuge at 10000r/min for 10 minutes, discard the supernatant, add 1 mL of 1 mol/L NaOH aqueous solution containing 10% (volume fraction) DMSO, seal the centrifuge tube with a sealing film, heat it in an 80°C water bath for 30 minutes, and measure the absorbance at 405nm. The inhibition rate of the sample on melanin synthesis was calculated according to formula (4): Melanin synthesis inhibition rate = (1-average absorbance of the sample group/average absorbance of the control group) × 100%.

Example 13

In vivo anti-aging activity evaluation

Experimental animal grouping, modeling and drug administration

1) 30 guinea pigs were randomly divided into a normal group (6) and an experimental group (24). The skin of about 3cm×3cm on the back was shaved for later use. The normal group was not given any special treatment; the experimental group was injected with progesterone at the flexor muscle at the base of the hind legs of the guinea pigs, 7.5mg/kg/d, once a day, alternating between the left and right legs, and the injection was continued for 30 days. At the same time, the exposed skin on the back was irradiated with medium-wave ultraviolet light (ultraviolet light from Shanghai Jiguang Special Lighting Appliance Co., Ltd.) with a wavelength of 280-320nm (the ultraviolet light was about 20cm away from the guinea pig's back), once a day, 60 minutes/time, for a total of 30 days.

2) Randomly select 6 guinea pigs in each experimental group and normal group, draw heart blood SOD and MDA test, take the full-thickness skin tissue of the back lesion of about 1.0cm×1.0cm, and perform HE and immunohistochemical staining. The remaining 18 guinea pigs in the experimental group were model groups, randomly divided into groups A, B, and C, with 6 in each group. Group A was treated and the skin reaction was closely observed. Group B applied vitamin E ointment on the lesion on the back of the chloasma mice, once a day; Group C did not receive any special treatment. Tests were performed one month later.

3) After the model was successfully established, 6 guinea pigs were randomly selected from the experimental group and the normal group, and SOD and MDA were detected by drawing cardiac blood. The full-thickness skin tissue of the skin lesion of about 1.0cm×1.0cm on the back was taken for HE and immunohistochemical staining. The remaining 18 guinea pigs in the experimental group were the model group, which were randomly divided into groups A, B, and C, with 6 in each group. Group A was not treated by the experimental group, and the skin reaction was closely observed. Vitamin E ointment was applied to the lesions on the back of the chloasma mice in group B once a day; group C was not treated with any special treatment, which was the blank group. The drugs were applied topically according to the proportion, once a day, and the medication was used continuously for 30 days. The blank group was topically applied with distilled water, once a day, and the time was the same as that of the experimental group. All guinea pigs were killed one day after the last administration, and 2 pieces of skin tissue were quickly taken from each guinea pig at the depilation site for use. After the tissue was processed, the MDA content was detected, as shown in Table 1, and the SOD activity was determined.

Table 1 MDA content detection

Experiments show that the leech extract can scavenge DPPH free radicals, has a strong ability to eliminate hydroxyl free radicals, and can effectively inhibit tyrosinase activity and inhibit the synthesis of melanin. Therefore, leech extract has an antioxidant effect; it can lighten chloasma and has the effect of whitening and lightening spots.

Embodiment 14

Stability test

For the accelerated test, the product was placed in a constant temperature and humidity chamber (temperature 40°C, relative humidity 75%) for 6 months, and samples were taken at 0, 1, 2, 3, and 6 months. For the long-term test, the product was placed in a constant temperature and humidity chamber (temperature 25°C, relative humidity 60%), and samples were taken at 0, 3, 6, and 12 months.

Table 2 Stability of hirudin extracts from Examples and Comparative Examples

Embodiment 15

Human safety evaluation test

The repetitive open smear test uses the flexion side of the forearm as the test site, with an area of 3× ^3cm2 . The test site should be kept dry and avoid contact with other topical preparations. Apply the test substance about 0.050 and 0.005g (mL)/time, twice a day, evenly to the test site for 7 consecutive days, and observe the skin reaction at the same time. If a skin reaction of 3 points or more occurs during this process, it should be decided whether to continue the test according to the specific situation. The skin reaction is observed according to the skin reaction evaluation criteria of the repetitive open smear test in Table 3, and the results are recorded.

Table 3 Evaluation criteria for skin reactions in repeated open smear tests

Table 4 Skin reaction scores in repeated open smear test

Group Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 score 0.03 0.02 0.03 0.01 0.01 0.04 Group Example 7 Example 8 Comparative Example 1 Comparative Example 2 Comparative Example 3 score 0.03 0.03 1.56 1.18 0.02

Table 5 Mask evaluation criteria

Sensory indicators describe Texture 1 to 10 points, the higher the score, the thicker the texture of the material odor 1 to 10 points, the higher the score, the more comfortable the smell Irritant 1 to 10 points, the higher the score, the less irritating Heavy 1 to 10 points, the higher the score, the lighter the material feels, and the skin feels less burdened

Table 6 Mask ratings

Group Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 score 7.54 7.69 8.01 7.95 8.56 7.58 Group Example 7 Example 8 Comparative Example 1 Comparative Example 2 Comparative Example 3 score 7.85 8.05 3.28 5.56 7.05

From the above feedback, it can be seen that the hirudin freeze-dried mask prepared by the present invention can avoid the hirudin from being inactivated too quickly in a liquid environment through the freeze-dried mask technology, which can not only prolong the storage time of the mask, but also maintain the activity of the mask. These results show that the hirudin freeze-dried mask prepared by the present invention has a good skin feel, good efficacy, and minimal irritation to the skin, providing comfortable and safe care for fragile skin.

Claims (8)

1. The hirudin freeze-dried mask is characterized by comprising a hirudin anti-aging freeze-dried mask and a hirudin whitening freeze-dried mask;

the hirudin anti-aging freeze-dried mask consists of active ingredients and auxiliary materials, wherein the active ingredients comprise: 0.2-8 parts of hirudin extract; the auxiliary materials comprise the following components: 1 to 99 parts of water, 1 to 50 parts of hydroxyethyl urea, 1 to 30 parts of sodium hyaluronate, 0.1 to 20 parts of jojoba seed oil, 0.25 to 10 parts of glycerin, 0.25 to 10 parts of trehalose, 0.2 to 10 parts of betaine, 0.1 to 8 parts of hydroxyethyl cellulose, 0.05 to 7 parts of fructo-oligosaccharide, 0.05 to 7 parts of collagen, 0.05 to 5 parts of squalane, 0.01 to 5 parts of polyglycerol-2 oleate and 0.01 to 2 parts of tremella fruit body extract;

the hirudin whitening freeze-dried mask consists of active ingredients and auxiliary materials, wherein the active ingredients comprise: hirudin extract, collagen, oligopeptide-10, oligopeptide-3, and allantoin; the auxiliary materials comprise the following components: mannitol, trehalose, sodium hyaluronate with molecular weight of 8000 dalton, sodium hyaluronate with molecular weight of 130 kilodalton, disodium hydrogen phosphate, sodium dihydrogen phosphate, glycerol and solvent.

2. The hirudin lyophilized mask of claim 1, wherein the hirudin extract comprises 3 parts, 50 parts of water, 20 parts of hydroxyethyl urea, 10 parts of sodium hyaluronate, 5 parts of jojoba seed oil, 3 parts of glycerin, 3 parts of trehalose, 3 parts of betaine, 1 part of hydroxyethyl cellulose, 0.8 part of collagen, 0.7 part of squalane, 0.3 part of polyglycerol-2 oleate, and 0.2 part of tremella fruit body extract.

3. The hirudin lyophilized mask of claim 1, wherein the hirudin extract is 1.2 parts, water is 70 parts, hydroxyethylurea is 9 parts, sodium hyaluronate is 6 parts, jojoba seed oil is 5 parts, glycerin is 2 parts, trehalose is 2 parts, betaine is 1.2 parts, fructo-oligosaccharide is 1.2 parts, hydroxyethylcellulose is 0.7 parts, fructo-oligosaccharide is 0.6 parts, collagen is 0.54 parts, squalane is 0.52 parts, polyglycerol-2 oleate is 0.03 parts, tremella fruit body extract is 0.01 parts.

4. The hirudin lyophilized mask of claim 1, wherein the hirudin extract is 1%, collagen is 1.5%, oligopeptide-10.5%, oligopeptide-30.5%, allantoin is 0.5%; mannitol 2.5%, trehalose 2.5%, sodium hyaluronate 0.25% with molecular weight 8000 daltons, sodium hyaluronate 0.25% with molecular weight 130 ten thousand daltons, disodium hydrogen phosphate 0.15%, sodium dihydrogen phosphate 0.12%, glycerin 2%, and solvent.

5. The hirudin lyophilized mask of claim 1, wherein the hirudin extract is 1.5%, collagen is 1.8%, oligopeptide-10.6%, oligopeptide-30.8%, allantoin is 0.6%; mannitol 3%, trehalose 2.5%, sodium hyaluronate 0.28% with molecular weight 8000 daltons, sodium hyaluronate 0.28% with molecular weight 130 ten thousand daltons, disodium hydrogen phosphate 0.25%, sodium dihydrogen phosphate 0.14%, glycerol 3%, and solvent.

6. The hirudin lyophilized mask of claim 1, wherein the hirudin extract is 2%, collagen is 2%, oligopeptide-11%, oligopeptide-31.5%, allantoin is 0.8%; mannitol 3%, trehalose 3.5%, sodium hyaluronate 0.35% with molecular weight 8000 daltons, sodium hyaluronate 0.29% with molecular weight 130 ten thousand daltons, disodium hydrogen phosphate 0.25%, sodium dihydrogen phosphate 0.15%, glycerin 4%, and solvent.

7. The hirudin lyophilized mask of claim 1, wherein the solvent is deionized water or purified water.

8. The preparation method of the hirudin freeze-dried mask is characterized by comprising the following steps of:

s1: dissolving the effective components in distilled water, and filtering with a 0.22 μm pore size filter membrane to obtain solution 1;

s2: dissolving auxiliary materials in deionized water to obtain a solution 2;

s3: mixing the solution 1 and the solution 2 to obtain a facial mask essence;

s4: sterilizing the mask cloth, folding, and immersing in mask essence; standing for 30-35 min until the mask cloth fully absorbs the mask essence;

s5: pushing the processed semi-finished product swing disc into a freeze dryer, rapidly cooling to-20 to-80 ℃, and keeping for 2-6 hours;

s6: after the base cloth and the material body are completely crystallized, vacuumizing is started to carry out sublimation drying; the sublimation drying time is 20-28 hours, and the air is discharged out of the box after the drying is finished;

s7: and rapidly filling the freeze-dried product into a mask packaging bag, carrying out cobalt radiation sterilization treatment in the mask packaging bag, vacuumizing the mask packaging bag in a nitrogen environment, and sealing to obtain the hirudin freeze-dried mask product.