CN117297996A

CN117297996A - Hirudin freeze-dried mask and preparation method thereof

Info

Publication number: CN117297996A
Application number: CN202310003683.5A
Authority: CN
Inventors: 尹俊林; 樊保敏; 王淇聿; 王阿英; 黄杰林; 徐建斌; 周永云; 曾广智; 陈景超; 郭亚飞; 王凯民; 和振秀; 孙蔚青; 李玖玲; 庞玉燕
Original assignee: Yunnan Minzu University
Current assignee: Yunnan Kaibei Technology Co.,Ltd.
Priority date: 2023-01-03
Filing date: 2023-01-03
Publication date: 2023-12-29

Abstract

The invention discloses a hirudin freeze-dried mask and a preparation method thereof, and experiments prove that the hirudin mask prepared by the preparation method of the hirudin freeze-dried mask has definite anti-aging and whitening effects; overcomes the defects that hirudin is unstable and easy to be deactivated in water, and has fishy smell at normal temperature; the fishy smell is purified and removed, and then the fishy smell is prepared into a freeze-dried mask for storage and use, so that the stability is improved, and the problem that the active ingredients added into cosmetics cannot be stored for a long time and the smell is solved; the freeze-dried mask provided by the invention has stable and effective activity, avoids the addition of unnecessary preservative and the like, and reduces the skin burden.

Description

Hirudin freeze-dried mask and preparation method thereof

Technical Field

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

Background

The facial mask is a facial skin cosmetic which is coated on the surface of human skin, is removed, scrubbed or reserved after a period of time, and has the functions of centralized cleaning, nursing and nutrition, and is popular with adults, especially female consumers. With the maturity of consumption psychology, people not only require the basic effects of cleaning, nursing and nutrition of the facial mask, but also further pursue the advanced effects of whitening, anti-aging and the like of the development facial mask. At present, the types of facial masks are various and are divided into a sticking facial mask, a tearing facial mask, a gel facial mask, a freeze-drying facial mask and the like. The freeze-dried mask is also called as freeze-dried powder mask and freeze-dried solid stock solution mask, and is formed by fusing components such as stock solution of the mask, a thickening agent, a humectant and the like with film cloth fibers, and sublimating moisture by vacuum freeze drying to form a solid dry film type mask. After the face is cleaned, the freeze-dried mask is directly taken out and applied on the face, the rest of the mask stays for about 20 minutes, and the rest of the mask is gently massaged and can be applied on hands and feet or other parts of the body. After the essence on the face is massaged and absorbed, the face is cleaned by clear water, and meanwhile, due to the good physical property of the freeze-dried mask, the freeze-dried mask can become a good medicine carrier, and different medicines are added into the freeze-dried mask, so that multiple effects of moisturizing and beautifying can be achieved.

Leech belongs to a highly specialized annelid animal, is originally carried in Shennong Ben Cao Jing (Shen nong's herbal), is commonly called Hirudo, and is a traditional Chinese medicine in China, salty in taste, bitter in taste and flat in nature. The pharmacopoeia of the people's republic of China has the curative effects of breaking blood, removing blood stasis and dredging channels, and is mainly used for treating tumor, masses, blood stasis, amenorrhea and traumatic injury. In 1884, haycraft first found a bioactive substance hirudin in the salivary glands of European medical leeches. In 1927, shionoya was first studied as an antithrombotic drug. 1955 Markwards indicates that it is a proteinaceous material and is designated hirudin. The natural hirudin is an anticoagulant protein extracted from medical Hirudo, and is a single-chain polypeptide composed of 65 or 66 amino acid residues, and has a relative molecular mass of 7000Dalton.

Hirudin itself has a strong fishy smell and is difficult to apply to skin care products; in the prior art, hirudin is applied to a mask, and is mainly used for a common patch mask and a smearing mask, which belong to a liquid environment, and a certain amount of preservative is required to be added. Part of human skin has irritation, allergy and other reactions to preservatives. Hirudin is a polypeptide which is also susceptible to inactivation and deterioration in a liquid environment.

Disclosure of Invention

In order to solve the technical problems, the invention provides a freeze-dried mask using hirudin as an active ingredient, which has definite anti-aging and whitening effects through experiments; overcomes the defects that hirudin is unstable and easy to be deactivated in water, and has fishy smell at normal temperature; the fishy smell is purified and removed, and then the fishy smell is prepared into a freeze-dried mask for storage and use, so that the stability is improved, and the problem that the active ingredients added into cosmetics cannot be stored for a long time and the smell is solved; the freeze-dried mask provided by the invention has stable and effective activity, avoids the addition of unnecessary preservative and the like, and reduces the skin burden.

In order to achieve the technical purpose, the invention is realized by the following technical scheme: a hirudin lyophilized facial mask comprises hirudin antiaging lyophilized facial mask and hirudin whitening lyophilized facial 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;

preferably, the hirudin extract comprises 3 parts of water 50 parts, 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;

preferably, 1.2 parts of hirudin extract, 70 parts of water, 9 parts of hydroxyethyl urea, 6 parts of sodium hyaluronate, 5 parts of jojoba seed oil, 2 parts of glycerin, 2 parts of trehalose, 1.2 parts of betaine, 1.2 parts of fructo-oligosaccharide, 0.7 part of hydroxyethyl cellulose, 0.6 part of fructo-oligosaccharide, 0.54 part of collagen, 0.52 part of squalane, 0.03 part of polyglycerol-2 oleate and 0.01 part of tremella fruit body extract;

preferably, the hirudin extract comprises 1% of hirudin extract, 1.5% of collagen, 10.5% of oligopeptide, 30.5% of oligopeptide and 0.5% of allantoin; 2.5% of mannitol, 2.5% of trehalose, 0.25% of sodium hyaluronate with molecular weight of 8000 daltons, 0.25% of sodium hyaluronate with molecular weight of 130 kilodaltons, 0.15% of disodium hydrogen phosphate, 0.12% of sodium dihydrogen phosphate, 2% of glycerol and a solvent;

preferably, the hirudin extract comprises 1.5%, collagen 1.8%, oligopeptide-10.6%, oligopeptide-30.8% and allantoin 0.6%; 3% of mannitol, 2.5% of trehalose, 0.28% of sodium hyaluronate with molecular weight of 8000 daltons, 0.28% of sodium hyaluronate with molecular weight of 130 kilodaltons, 0.25% of disodium hydrogen phosphate, 0.14% of sodium dihydrogen phosphate, 3% of glycerol and a solvent;

preferably, the hirudin extract is 2%, collagen is 2%, oligopeptide-11%, oligopeptide-31.5%, and allantoin is 0.8%; 3% of mannitol, 3.5% of trehalose, 0.35% of sodium hyaluronate with molecular weight of 8000 daltons, 0.29% of sodium hyaluronate with molecular weight of 130 kilodaltons, 0.25% of disodium hydrogen phosphate, 0.15% of sodium dihydrogen phosphate and 4% of glycerin;

preferably, the solvent is deionized water or purified water.

The invention also aims to provide a preparation method of the hirudin freeze-dried mask, which comprises the following steps:

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.

The beneficial effects of the invention are as follows:

compared with the prior art, the hirudin freeze-dried mask provided by the invention overcomes the defect that functional components such as peptides are easy to inactivate in a liquid environment; the preservative must be added in the liquid form, so that the skin is irritated and allergic; and product ingredients in liquid form and instability problems; the active raw materials of the product are stable and effective, so that unnecessary addition of preservatives and the like is avoided, and the product is more friendly to the skin; experiments prove that the cream has definite anti-aging and whitening effects.

Drawings

FIG. 1 is a graph showing the comparison of anticoagulation activity;

FIG. 2 is a graph of the results of fishy smell sensory scores;

FIG. 3 is a graph showing the comparison of the free radical elimination rates of hirudin and VC DPPH;

FIG. 4 is a graph showing the comparison of the rate of radical elimination of hydroxyl groups between hirudin and VC;

FIG. 5 is the effect of hirudin and arbutin on B16 intracellular tyrosinase activity;

FIG. 6 is the effect of hirudin and arbutin on melanin synthesis in B16 cells;

FIG. 7 is a diagram showing the result of the construction of the chloasma model;

FIG. 8 is a graph of the results of HE dye verification modeling success for the normal and model sets;

fig. 9 is a graph showing the result of the change of back skin lesions by adding ultraviolet irradiation to progesterone administration for 30 days;

FIG. 10 is a graph showing the results of gray scale ratio after administration;

FIG. 11 is a graph showing MDA content versus results.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

In this example, raw materials were weighed out sequentially, 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 glycerin, 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 fructo-oligosaccharide, 0.05 kg of collagen, 0.032 kg of squalane, 0.021 kg of polyglycerol-2 oleate, and 0.01 kg of tremella fruit body extract group. The freeze-dried mask product is prepared according to the following steps:

the raw materials weighed above are prepared into a freeze-dried mask according to the following steps:

s1: preparing water solution of 4 kg of water, hydroxyethyl urea, sodium hyaluronate, jojoba seed oil, glycerol, trehalose, betaine, hydroxyethyl cellulose, fructo-oligosaccharide, collagen, squalane, polyglycerol-2 oleate, tremella fruit body extract group and other raw materials according to a proportion to obtain solution 1;

s2: dissolving hirudin in the residual water, and filtering with a filter membrane with pore diameter of 0.22 μm to obtain solution 2;

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

s4: sterilizing the mask cloth, and well folding and soaking the mask cloth in mask essence according to a fixed folding method;

s5: standing for 30 minutes until the mask base cloth fully absorbs the full material;

s6: pushing the processed semi-finished product swing disc into a freeze dryer, rapidly cooling to-20 ℃, and keeping for 2 hours;

s7: after the base cloth and the material body in the bottle are completely crystallized, vacuum pumping is started to carry out sublimation drying at 23 pa;

s8: the sublimation drying time is 21 hours, and the air is discharged out of the box after the drying is finished;

s9: and rapidly filling the freeze-dried product into a mask packaging bag, sterilizing the mask packaging bag, vacuumizing the mask packaging bag in a nitrogen environment, and sealing to obtain the hirudin freeze-dried mask product.

Example 2

In this example, raw materials were weighed out in order, 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 glycerin, 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 fructo-oligosaccharide, 0.1 kg of collagen, 0.03 kg of squalane, 0.01 kg of polyglycerol-2 oleate, and 0.01 kg of tremella fruit body extract group. The freeze-dried mask product is prepared according to the following steps:

the freeze-dried mask was prepared according to the method of example 1, except that the freeze dryer was rapidly cooled to-30 ℃ for 2.5 hours; vacuum was applied to the mixture at 30pa and sublimation drying was performed for 25 hours.

Example 3

In this example, raw materials were weighed out in order, 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 glycerin, 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 fructo-oligosaccharide, 0.07 kg of collagen, 0.05 kg of squalane, 0.015 kg of polyglycerol-2 oleate, and 0.015 kg of tremella fruit body extract group. The freeze-dried mask product is prepared according to the following steps:

the freeze-dried mask was prepared according to the method of example 1, except that the freeze dryer was rapidly cooled to-40 ℃ for 1.9 hours; sublimation drying was performed for 18 hours by vacuum 45 pa.

Example 4

In this example, raw materials were weighed out sequentially, 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 glycerin, 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 fructo-oligosaccharide, 0.06 kg of collagen, 0.05 kg of squalane, 0.012 kg of polyglycerol-2 oleate, and 0.011 kg of tremella fruit body extract group. The freeze-dried mask product is prepared according to the following steps:

the freeze-dried mask was prepared according to the method of example 1, except that the freeze dryer was rapidly cooled to-45 ℃ for 2.3 hours; sublimation drying was performed for 23 hours by evacuating 48 pa.

Example 5

In this example, raw materials were weighed out sequentially, 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 glycerin, 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 fructo-oligosaccharide, 0.041 kg of collagen, 0.032 kg of squalane, 0.006 kg of polyglycerol-2 oleate, and 0.005 kg of tremella fruit body extract group. The freeze-dried mask product is prepared according to the following steps:

the freeze-dried mask was prepared according to the method of example 1, except that the freeze dryer was rapidly cooled to-48 ℃ for 1.8 hours; vacuum was applied at 50pa and sublimation drying was performed for 30 hours.

Example 6

In this embodiment, the raw materials of efficacy, collagen, oligopeptide-1, oligopeptide-3 and hirudin are prepared in sequence, and the auxiliary materials comprise the following components according to the final concentration: mannitol, trehalose, sodium hyaluronate with molecular weight of 8000 dalton, sodium hyaluronate with molecular weight of 130 kilodalton, disodium hydrogen phosphate, sodium dihydrogen phosphate and glycerol, and the solvent is deionized water.

The freeze-dried mask product is prepared according to the following steps:

the raw materials prepared above are prepared into a freeze-dried mask according to the following steps:

s1: dissolving collagen, oligopeptide-1, oligopeptide-3 and hirudin in water according to a certain proportion to obtain a solution A;

s2: filtering the solution A by using a filter membrane with the pore diameter of 0.22 micron to obtain a solution 1;

s3: mixing part of deionized water with mannitol, trehalose, sodium hyaluronate with molecular weight of 8000 daltons, sodium hyaluronate with molecular weight of 130 kilodaltons, disodium hydrogen phosphate and sodium dihydrogen phosphate to obtain facial mask essence;

Example 7

In this example, raw materials for efficacy, collagen 1.5%, oligopeptide-10.5%, oligopeptide-30.6% and hirudin 1.5% were prepared in sequence, and the auxiliary materials comprise, according to the composition of the final concentration: mannitol 2.5%, trehalose 2.5%, sodium hyaluronate 0.25% with molecular weight 8000 daltons, sodium hyaluronate 15% with molecular weight 130 ten thousand daltons, disodium hydrogen phosphate 0.14%, sodium dihydrogen phosphate 0.12%, glycerin 2%, and partially deionized water.

The freeze-dried mask product is prepared according to the following steps:

the freeze-dried mask was prepared according to the method of example 1, except that the freeze dryer was rapidly cooled to-30 ℃ and held for 3 hours; vacuum was applied to the mixture at 30pa and sublimation drying was performed for 25 hours.

Example 8

In this example, raw materials for efficacy, collagen 2.5%, oligopeptide-11.5%, oligopeptide-30.8% and hirudin 1.8% were prepared in sequence, and the auxiliary materials comprise, according to the composition of the final concentration: mannitol 2.8%, trehalose 2.5%, sodium hyaluronate 0.45% with molecular weight 8000 daltons, sodium hyaluronate 0.15% with molecular weight 130 ten thousand daltons, disodium hydrogen phosphate 0.18%, sodium dihydrogen phosphate 0.15%, glycerol 3%, and partially deionized water.

The freeze-dried mask product is prepared according to the following steps:

the freeze-dried mask was prepared according to the method of example 1, except that the freeze dryer was rapidly cooled to-40 ℃ for 2 hours; vacuum 45pa was applied to sublimation dry for 20 hours.

Example 9

Comparative example 1

The difference from the mask prepared in example 1 and example 6 described above is that comparative example 1 was not subjected to lyophilization treatment, and the remaining component contents and preparation methods were the same as in example 1 and example 6. Hirudin is unstable in water, has bad taste after 7 days and has reduced activity.

Comparative example 2

The mask prepared in example 1 and example 6 was different from the mask prepared in example 1 and example 6 in that the hirudin solution was not filtered, and the content of the remaining components and the preparation method of comparative example 2 were as described in example 1 and example 6. The bacterial content of the product cannot be guaranteed to be in compliance by filtration and sterilization.

Comparative example 3

The mask produced in example 1 and example 6 was different from the mask produced in example 1 and example 6 in that the solution 1 was sterilized at high temperature, and the content of the remaining components and the preparation method were as described in example 1 and example 6. The hirudin is deactivated after high temperature sterilization.

Example 10

Hirudin deodorization process

Extracting hirudin at a feed liquid ratio of 20:1g/ml, performing ultrasonic treatment for 30min for three times, mixing the extractive solutions, performing deodorization treatment on the combined supernatant, performing freeze-drying, and then performing measurement on the hirudin. Titration was performed with 40NIH/mL thrombin solution and 20NIH/mL thrombin solution was used near the endpoint. The thrombin volume was 1-5. Mu.L and titrated with a titration volume of 1. Mu.L near the endpoint. The volume of the thrombin solution is 5 mu L each time, the thrombin is stirred by a syringe needle once each time, and the thrombin solution is titrated every 1min and is finished within 10min when the time is close to the time limit. Calculating activity; sample solution activity = Σthrombinconcentration x drop volume, thereby converting the sample hirudin content. As shown in fig. 1, 1 is the original, 2 is the anticoagulation activity after water extraction treatment, 3 to 8 are the anticoagulation activities after different deodorizing methods, and 3 has the best anticoagulation effect compared with 2, and 2 and 3 have significant differences compared with 1 (P < 0.05).

Finally, the test was sensory rated by a panel of 10 panelists (22-26 years old, 5 men and women each). The fishy smell evaluation of the experiment adopts 10 minutes, the distilled water without fishy smell is 0 minutes, the untreated hirudin is 10 minutes, the sample is put into a tube during evaluation, and an evaluator performs sensory evaluation according to the evaluation rule and finally takes the average value as the final score. The score is 0-2, and no fishy smell is basically generated; 2-4, little fishy smell; 4 to 6, fishy smell; 6-8: the fishy smell is obvious; 8-10, strong fishy smell. And screening the optimal fishy smell removing process by taking antithrombin activity and sensory evaluation as indexes. The hirudin antithrombin activity treated by the method is 511.1ATU/g, and the sensory evaluation score is 3 minutes. The conclusion is that the purification method 3 has the best fishy smell removing effect on the hirudin, and simultaneously the antithrombin activity is preserved to the greatest extent. As shown in FIG. 2, the original form is water extraction treatment, the deodorizing effect of 2-8 different deodorizing effects is the best compared with the original form, and the method for purifying the deodorizing effect of 2 is determined by combining the anticoagulation effect of 2.

Example 11

Determination of antioxidant Activity in vitro

Determination of DPPH radical scavenging ability

Mask samples with mass concentrations of 5, 2.5, 1.25, 0.625 and 0.3125mg/mL were prepared with distilled water. Taking 0.7mL of samples with different mass concentrations, adding 1.3mL of 0.3mmol/L DPPH ethanol solution, and measuring the absorbance value of the mixture at the wavelength of 517nm after the light-shielding reaction is completed, wherein VC is used as a reference; as shown in fig. 3;

wherein: a0 is a blank control absorbance value, A1 is a sample solution absorbance value, and A2 is an absolute ethyl alcohol and sample solution absorbance value;

determination of the ability to scavenge hydroxyl radicals

Mask samples with mass concentrations of 5, 2.5, 1.25, 0.625 and 0.3125mg/mL were prepared with distilled water. Adding 0.2mL of a ferrous sulfate solution and a salicylic acid-ethanol solution with the concentration of 6mmol/L into a reaction system in sequence, adding 0.2mL of a hydrogen peroxide solution with the concentration of 0.2mL of each sample solution with the concentration of 0.2mL of 6mmol/L, reacting for 1h at 37 ℃, and measuring the absorbance value of the mixture at the wavelength of 510nm after the reaction is completed. As shown in fig. 4;

wherein: a0 is a blank control absorbance value, A1 is a sample absorbance value, and A2 is a sample background absorption without salicylic acid-ethanol solution.

Example 12

1) Determination of tyrosinase activity in B16 cells

Culturing in 96-well plate, adding B16 single cell suspension (7-8). Times.10 ³ After 24h of adherence, adding culture medium containing samples with different mass concentrations, wherein 3 compound holes are arranged in each mass concentration. The control group replaced the sample solution with RPMI-1640 complete medium. 37 ℃,5% CO ₂ After incubation for 72h under conditions, the supernatant was discarded. Washing with PBS (pH=7.4) for 2 times, adding 50 μl of 1% octyl phenol polyoxyethylene ether (Triton X-100) solution into each well, rapidly freezing at-80deg.C for 30min, thawing at room temperature to completely rupture cells, pre-warming at 37deg.C for 5min, adding 10 μl of 1% L-DOPA solution, reacting at 37deg.C for 2 hr, and adding enzymeThe absorbance was measured at 490 nm. Tyrosinase activity inhibition was calculated as inhibition=1- (average absorbance of sample group/average absorbance of control group) ×100%.

2) Determination of melanin content in B16 cells

B16 cells in logarithmic growth phase were grown at a density of 5×10 ⁴ Each of the cells was inoculated into a 6-well plate at a volume of 2mL per well, and cultured for 24 hours in a conventional manner. The culture solutions of samples with different mass concentrations are replaced, the control group is added with fresh complete culture medium with the same volume, and 3 parallel compound holes are arranged in each group. At 37℃with 5% CO ₂ After culturing at saturated humidity for 72 hours, the supernatant was discarded. Each well is washed once by PBS, 0.5mL of trypsin digestion liquid with the mass concentration of 5g/L is added into the culture box at 37 ℃ for digestion for 2 to 4 minutes, and the whole digestion of cells is ensured by observation under a microscope. Digestion was stopped by adding 1mL of RPMI-1640 complete medium, gently beating the cells and collecting them into a 1.5mL centrifuge tube, centrifuging at 10000r/min for 10min, discarding the supernatant, adding 1mL of 1mol/LNaOH aqueous solution containing 10% (volume fraction) DMSO, sealing the centrifuge tube with a sealing film, heating in 80℃water bath for 30min, and measuring absorbance at 405 nm. The inhibition ratio of the sample to melanin synthesis was calculated according to formula (4). Melanin synthesis inhibition = (1-sample group average absorbance/control group average absorbance) ×100%.

Example 13

Evaluation of anti-aging Activity in vivo

Grouping, modeling and administration of experimental animals

1) 30 guinea pigs were randomly divided into a normal group (6) and an experimental group (24), and skin shaves Mao Beiyong were performed on the back of about 3cm×3 cm. The normal group is not specially treated; the experimental group is to inject progesterone into the root of the back leg of a guinea pig, 7.5mg/kg/d,1 d/time, alternate left and right legs, continuously inject the mould for 30d, and simultaneously irradiate the back exposed skin (the ultraviolet lamp is about 20cm from the back of the guinea pig) with medium wave ultraviolet rays (ultraviolet lamp of Shanghai Ji Guang special illumination electric appliances Co., ltd.) with the wavelength of 280-320 nm every day, 1 time/d, 60 min/time and 30d.

2) 6 guinea pigs in the experimental group and the normal group are randomly selected, heart blood is extracted for SOD and MDA detection, and the whole skin tissue at the skin lesion position of about 1.0cm multiplied by 1.0cm at the back is taken for HE and immunohistochemical staining. The experimental groups were divided into A, B, C groups at random, with 6 groups each, with the remaining 18 model groups. After the group A is treated and skin reaction is closely observed, the group B is smeared with vitamin E ointment at the back focus skin lesion of the chloasma mice for 1 time/d; group C did not make any special treatment. The test was performed after 1 month.

3) 6 guinea pigs of the experimental group and the normal group are randomly selected after the molding is successful, the heart blood is extracted for SOD and MDA detection, and the whole skin tissue at the skin lesion position of about 1.0cm multiplied by 1.0cm at the back is taken for HE and immunohistochemical staining. The experimental groups were divided into A, B, C groups at random, with 6 groups each, with the remaining 18 model groups. The group A is treated by a non-experimental group, and after skin reaction is closely observed, the group B is smeared with vitamin E ointment at the lesion skin of the back part of the chloasma mouse for 1 time/d; group C was blank without any special treatment. The local smearing medicine is calculated according to the proportion for 1 time/d and the medicine is continuously used for 30 days. The blank groups were applied with distilled water locally for 1 time/d for the same time as the experimental groups. All guinea pigs were sacrificed 1 day after the last dose, and each guinea pig was promptly harvested at the dehairing site for 2 pieces of skin tissue for later use. MDA content detection is carried out after the tissue is treated, and is shown in Table 1, and SOD activity is measured.

TABLE 1MDA content detection

Experiments show that the Hirudo manillensis extract has the effects of eliminating DPPH free radical, and has strong capability of eliminating hydroxyl free radical, and can effectively inhibit tyrosinase activity and inhibit melanin synthesis, so that the hirudin extract has an antioxidation effect; can lighten chloasma, and has the effects of whitening and lightening chloasma.

Example 14

Stability test

The products are placed in a constant temperature and humidity box (the temperature is 40 ℃ and the relative humidity is 75%) for 6 months, and sampling and detection are carried out respectively at 0 month, 1 month, 2 month, 3 month and 6 month. Long-term test the products were placed in a constant temperature and humidity cabinet (temperature 25 ℃, relative humidity 60%) and sampled and measured at 0, 3, 6, 12 months.

Table 2 stability of hirudin extract of examples and comparative examples

/>

Example 15

Human safety evaluation test

The repeated open type smearing test uses forearm flexor side as tested part, and the area is 3×3cm ² The tested part should be kept dry to avoid contacting with other external preparations. The test object is uniformly applied to the test site about 0.050, 0.005g (mL)/time, 2 times daily for 7 consecutive days while observing skin reaction, and whether to continue the test should be determined according to circumstances during the course of such skin reaction as occurrence of 3 minutes or more. Skin reactions were observed according to the skin reaction evaluation criteria of the repeated open-type smear test of table 3, and the results were recorded.

TABLE 3 skin reaction evaluation criteria for open-type skin repeatability application test

TABLE 4 skin response score for skin repetitive open spread test

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

TABLE 5 mask evaluation criteria

Sensory index	Description of the invention
		Texture of texture	1-10 min, the higher the fraction, the thicker the texture of the material
Smell of	1-10 min, the higher the score, the more comfortable the smell sense
		Irritation (irritation)	1-10 min, the higher the fraction, the less irritating
Sense of heaviness	1 to 10 minutes, the higher the fraction, the lighter the sensation of the body, and the no burden on the skin

Table 6 mask scoring

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

According to the feedback, the hirudin freeze-dried mask prepared by the invention can avoid the over-quick inactivation of the hirudin in a liquid environment by the freeze-dried mask technology, so that the storage time of the mask can be prolonged, and the activity of the mask can be maintained. The results show that the hirudin freeze-dried mask prepared by the invention has good skin feel, good efficacy and little skin irritation, and gives comfortable and safe care to fragile skin.

Claims

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 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:

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;