CN116270287A - Anti-aging and ultraviolet injury-resistant hirudin extract freeze-dried essence and production process thereof - Google Patents

Abstract

The invention discloses an anti-aging ultraviolet injury-resistant hirudin extract freeze-dried essence and a preparation method thereof, wherein the freeze-dried essence consists of freeze-dried powder and solvent liquid; the freeze-dried powder comprises: hirudin extract, mannitol, oligopeptide-3, ceramide, trehalose and arginine; the solvent liquid comprises: glycerol, propylene glycol, hydrolyzed sodium hyaluronate, dipotassium glycyrrhizinate, hexapeptide-9, butanediol, squalane, PEG castor oil, hydrolyzed collagen, nicotinamide and water; the water quality extract freeze-dried essence prepared by the method provided by the invention has the advantages that the fishy smell is removed, and the activity of the water quality extract freeze-dried essence is still kept; the freeze-drying technology can avoid the inactivation of the hirudin in a liquid environment, so that the stability is improved, the activity is reserved, and the hirudin is favorable for long-term storage and use; the active raw materials of the product are stable and effective, the addition of unnecessary preservative and the like is avoided, and the damage of the preservative to the skin can be avoided while the anti-aging and anti-ultraviolet injury are realized.

Description

Anti-aging and ultraviolet injury-resistant hirudin extract freeze-dried essence and production process thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to an anti-aging ultraviolet injury-resistant hirudin extract freeze-dried essence and a production process thereof.

Background

Continued exposure of the skin to ultraviolet radiation can lead to ultraviolet damage characterized by structural and functional abnormalities of the skin tissue. Morphologically, uv damage induces the occurrence and exacerbation of skin tissue damage, roughness, deep wrinkles and pigmentation; in physiological aspects, a great deal of evidence shows that ultraviolet irradiation can stimulate the release of Reactive Oxygen Species (ROS) and inflammatory cytokines, finally causes oxidation stress reaction of human skin, causes wrinkles and tissue fragility to increase, promotes skin injury and immune dysfunction, increases the probability of cell injury, further induces a series of proliferative lesions, and can lead to squamous cell carcinoma, malignant melanoma and the like in serious cases.

In modern cities, skin damage caused by sun exposure, radiation and the like is ubiquitous, so that various skin problems are caused, people gradually realize that ultraviolet radiation can cause photo-aging of the skin, collagen loss and water loss are caused, and a plurality of skin problems are caused, so that the awareness of skin protection by using cosmetics for preventing ultraviolet and aging is also increasing. At present, the medicines for clinically treating skin ultraviolet injury are mainly vitamin A acid medicines, and the most common adverse reaction is dry skin mucosa and has teratogenic effect. Therefore, the active search and development of the skin external ultraviolet injury treatment medicine with good curative effect and few side effects has great clinical value and demand potential. The essence is one of skin care products for the face, is suitable for adding various functional components with higher concentration, has small skin irritation, is suitable for being used as a carrier of skin care substances with anti-ultraviolet and anti-aging functions, and is natural, pollution-free and high-safety extract which becomes one of research hotspots in anti-ultraviolet injury and aging cosmetics.

Hirudin is a substance secreted by the salivary glands of blood sucking leeches, is a single-chain polypeptide consisting of 65-66 amino acid residues, and is an intermediate substance consisting of amino acids but different from protein. It has the characteristics of protein, but is simpler than protein, has less molecular weight and is easy to be absorbed by organisms. Wei Shuyi, yan Guo, han Zhijiang, et al, the "natural hirudin hydrogel improved the survival of the random skin flap" (Chinese tissue engineering research, 2013,17 (53): 9107-9112.) showed that natural hirudin gel was permeable into intact skin tissue, did not show any irritation to intact skin and damaged skin of Wistar rats, and did not show toxicity to skin when used in large doses in the short term. Wei Guili the hirudin mentioned in the "Hirudo extract has good antioxidant effect on melanoma cells, and antioxidant activity and safety evaluation" has good antioxidant effect in vitro. Chinese patent CN201210559842.1 discloses hirudin whitening and moisturizing conditioning cream which can independently and directly permeate into dermis of skin and is easily absorbed by the skin.

However, the prior art use of hirudin has the following drawbacks:

1) In the prior art, hirudin has certain fishy smell, so that the application of the hirudin in cosmetics is limited;

2) The hirudin has low stability in water and is easy to inactivate and deteriorate;

3) Based on the two defects, hirudin is not applied to skin for resisting aging and ultraviolet injury at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides an anti-aging ultraviolet injury-resistant hirudin extract freeze-dried essence and a production process thereof, and the technical scheme of the invention overcomes the defects that hirudin has fishy smell and is easy to inactivate and deteriorate in water; the freeze-dried essence prepared by purifying and removing the fishy smell is stored in the form of freeze-dried powder, so that the stability is improved, the problem that the active ingredients added into cosmetics cannot be stored for a long time and the smell is solved, and the prepared freeze-dried essence has good effects of resisting ultraviolet and resisting aging.

In order to achieve the technical purpose, the invention is realized by the following technical scheme:

a freeze-dried essence of hirudin extract for resisting aging and ultraviolet injury comprises freeze-dried powder and solvent solution;

the freeze-dried powder comprises: hirudin extract, mannitol, oligopeptide-3, ceramide, trehalose and arginine;

the solvent liquid comprises: glycerol, propylene glycol, hydrolyzed sodium hyaluronate, dipotassium glycyrrhizinate, hexapeptide-9, butanediol, squalane, PEG castor oil, hydrolyzed collagen, nicotinamide and water;

the freeze-dried powder comprises, by mass, 1% -2% of hirudin extract, 4% -5% of mannitol, 31.5% -2% of oligopeptide, 0.5% -1% of ceramide, 0.8% -1% of trehalose, 0.08% -1% of arginine and the balance deionized water; the solvent liquid comprises the following components in percentage by mass: 8-10% of glycerol, 7-8% of propylene glycol, 1-2% of hydrolyzed sodium hyaluronate, 2-3% of dipotassium glycyrrhizinate, 91-2% of hexapeptide, 1-1.5% of butanediol, 8-9% of squalane, 7-8% of PEG castor oil, 1-2% of hydrolyzed collagen, 0.4-0.5% of nicotinamide and the balance of deionized water;

preferably, the freeze-dried powder comprises the following components in percentage by mass: 1% of hirudin extract, 3% of mannitol, 31% of oligopeptide, 0.5% of ceramide, 0.8% of trehalose, 0.05% of arginine and the balance of deionized water; the solvent liquid comprises the following components in percentage by mass: 6% of glycerin, 7% of propylene glycol, 0.8% of hydrolyzed sodium hyaluronate, 1% of dipotassium glycyrrhizinate, 90.5% of hexapeptide, 0.6% of butanediol, 5% of squalane, 5% of PEG castor oil, 0.5% of hydrolyzed collagen, 0.2% of nicotinamide and the balance of deionized water;

preferably, the freeze-dried powder comprises the following components in percentage by mass: 2% of hirudin extract, 5% of mannitol, 32% of oligopeptide-32%, 0.8% of ceramide, 1% of trehalose, 1% of arginine and the balance of deionized water; the solvent liquid comprises the following components in percentage by mass: 10% of glycerol, 8% of propylene glycol, 1% of hydrolyzed sodium hyaluronate, 2% of dipotassium glycyrrhizinate, 92% of hexapeptide, 1.5% of butanediol, 8% of squalane, 7% of PEG castor oil, 1% of hydrolyzed collagen, 0.4% of nicotinamide and the balance of deionized water;

the invention also aims at providing a preparation method of the freeze-dried hirudin extract essence for resisting aging and ultraviolet injury, which comprises the following steps:

s1: dissolving hirudin in water to obtain solution, purifying, deodorizing, and filtering with 0.22 μm filter membrane to obtain hirudin extract;

s2: preparing a solution from raw materials such as hirudin extract, mannitol, oligopeptide-3, ceramide, trehalose, arginine, the balance deionized water and the like according to a proportion;

s3: shaking the solution uniformly, filtering with a 0.22 μm filter membrane to obtain solution A, standing for over 36h, and canning;

s4: vacuum freeze-drying the canned solution A to obtain freeze-dried powder;

s5: dissolving glycerol, propylene glycol, hydrolyzed sodium hyaluronate, dipotassium glycyrrhizinate, hexapeptide-9, butanediol, squalane, PEG castor oil, hydrolyzed collagen, nicotinamide and the balance deionized water in proportion, and sterilizing in an autoclave at constant temperature of 121 ℃ for 30min to obtain a solution B; standing for more than 24 hours, and canning;

s6: the ratio of the freeze-dried powder to the essence is 50mg:3mL of freeze-dried powder and solvent liquid are uniformly mixed to obtain the hirudin extract freeze-dried essence.

The beneficial effects of the invention are as follows:

the water quality extract freeze-dried essence prepared by the method provided by the invention has the advantages that the fishy smell is removed, and the activity of the water quality extract freeze-dried essence is still kept; the freeze-drying technology can avoid the inactivation of the hirudin in a liquid environment, so that the stability is improved, the activity is reserved, and the hirudin is favorable for long-term storage and use; the active raw materials of the product are stable and effective, the addition of unnecessary preservative and the like is avoided, and the damage of the preservative to the skin can be avoided while the anti-aging and anti-ultraviolet injury are realized.

Drawings

FIG. 1 is a graph showing comparison of DPPH radical elimination rate results;

FIG. 2 is a graph comparing the results of hydroxyl radical elimination;

FIG. 3 is a graph comparing the results of superoxide radical elimination rate;

FIG. 4 is a diagram showing the experimental procedures of grouping, modeling and administration of mice experimental animals;

FIG. 5 is a bar graph of the effect of moisture content on the skin of mice;

FIG. 6 is a graph of the results of HE staining of mouse skin tissue;

FIG. 7 is a schematic representation of the results of Masson staining of mouse skin tissue;

FIG. 8 is a schematic representation of the results of changes in antioxidant enzyme activity in the skin of mice.

Detailed Description

The following description will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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 1, raw materials were prepared in sequence, and hirudin extract was prepared first, and hirudin was mixed in a feed ratio of 1: dissolving 20g/ml in water, extracting the water solution, purifying, deodorizing, and vacuum freeze drying to obtain hirudin extract. The other raw materials are prepared from the following components in parts by weight: 1% of hirudin extract, 6% of mannitol, 1.2% of trehalose, 30.000085% of oligopeptide, 0.08% of ceramide, 0.6% of arginine and the balance of deionized water.

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

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

s1: after being uniformly mixed, each component is canned in a penicillin bottle, is placed in a freeze-drying bin partition plate, enters a pre-cooling stage of a front box, and a circulating pump is selectively started according to a set; and then the first compressor and the corresponding plate cooling valve are started, and when the temperature of the product reaches-80 ℃, the rear box is refrigerated. When the temperature of the coil pipe of the rear box reaches-80 ℃, the pre-vacuumizing is started for 2.5 hours, the sublimation control is started, the electric heating is started, the cold mixing valve is closed, the temperature is firstly increased to-50 ℃ for 5 hours, the temperature is increased to-25 ℃ for 3 hours, the temperature is increased to-10 ℃ for 2 hours, the temperature is increased to 0 ℃ for 3 hours, the temperature is increased to 15 ℃ for 2 hours, the temperature is increased to 28 ℃ for 5 hours, and freeze-drying is finished, so that the freeze-dried powder can be obtained.

S2: preparation of a solvent liquid: weighing the following components in percentage by mass: 5% of glycerol, 4% of propylene glycol, 0.3% of hydrolyzed sodium hyaluronate, 2% of dipotassium glycyrrhizinate, 92% of hexapeptide, 1.5% of butanediol, 2.5% of squalane, 0.08% of PEG castor oil, 0.55% of hydrolyzed collagen, 0.26% of nicotinamide and the balance of deionized water. Mixing glycerol, propylene glycol, nicotinamide and PEG castor oil, adding water at 500r/min at room temperature, stirring for 30min, heating to 80deg.C after stirring, maintaining the temperature for 30min, cooling to 40deg.C, adding hirudin extract, dipotassium glycyrrhizinate, hexapeptide-9, squalane and hydrolyzed collagen, and stirring for 30 min.

S3: the prepared freeze-dried powder and solvent liquid are mixed according to the feed liquid ratio of 50mg:3mL of the mixture is mixed to obtain the freeze-dried essence with the functions of resisting ultraviolet injury and aging.

Example 2

Based on the example 1, the difference is that the following components are weighed according to mass percent in the preparation process of the freeze-dried powder: 1.5% of hirudin extract, 4% of mannitol, 2% of trehalose, oligopeptide-30.002%, 1% of ceramide, 0.05% of arginine and the balance of deionized water; the solvent liquid is prepared by weighing the following components in percentage by mass: 10% of glycerin, 2% of propylene glycol, 0.5% of dipotassium glycyrrhizinate, 90.5% of hexapeptide, 2% of butanediol, 0.04% of hydrolyzed collagen, 0.5% of nicotinamide, 0.1% of PEG castor oil and the balance of deionized water.

Example 3

Based on the example 1, the difference is that the following components are weighed according to mass percent in the preparation process of the freeze-dried powder: 1.8% of hirudin extract, 5% of mannitol, 0.05% of trehalose, 30.00003% of oligopeptide, 0.01% of ceramide, 1% of arginine and the balance of deionized water; the solvent liquid is prepared by weighing the following components in percentage by mass: 4% of glycerol, 8% of propylene glycol, 3% of dipotassium glycyrrhizinate, 91.5% of hexapeptide, 2% of butanediol, 1% of hydrolyzed collagen, 0.35% of nicotinamide, 0.2% of PEG castor oil and the balance of deionized water.

Example 4

Based on the example 1, the difference is that the following components are weighed according to mass percent in the preparation process of the freeze-dried powder: 1.8% of hirudin extract, 5% of mannitol, 0.05% of trehalose, 30.00003% of oligopeptide, 0.01% of ceramide, 1% of arginine and the balance of deionized water; the solvent liquid is prepared by weighing the following components in percentage by mass: 4% of glycerol, 8% of propylene glycol, 3% of dipotassium glycyrrhizinate, 91.5% of hexapeptide, 2% of butanediol, 1% of hydrolyzed collagen, 0.35% of nicotinamide, 0.2% of PEG castor oil and the balance of deionized water.

Example 5

Comparative experiment setup

Comparative example 1 is identical to example 1, except that no hirudin extract is added;

comparative example 2 is identical to example 1, except that no oligopeptide-3 component is added;

comparative example 3 is the same as example 1 except that no ceramide component is added;

comparative example 4 was the same as example 1 except that the trehalose component was not added;

experimental procedure

Taking the essence obtained in examples 1-4 and comparative examples 1-4, preparing samples, respectively detecting the performances of the samples and recording the detection results;

1. acute oral toxicity test: the method is characterized in that 8 groups of Kunming mice with the weight of 18-22g are respectively arranged in male and female halves, 10 mice in each group are subjected to test at the temperature of 20-24 ℃ and the humidity of 60-70%, the fasted time is 16 hours before the administration of the mice, the mice are basically normal in activity and diet after orally taking 5000mg/kg (0.5 ml/20g of body weight) of raw water solution at a time, the body weight is normal after continuous observation for 7 days, obvious toxic reaction and death of the animals do not occur, and the essence prepared by the method is safe and nontoxic.

2. Anti-wrinkle anti-aging experiment

1) Measurement of DPPH radical scavenging ability hirudin samples with mass concentrations of 10, 5, 2.5, 1.25, 0.625mg/mL were prepared with distilled water. Samples of different mass concentrations were taken, 0.7mL each, 1.3mL of 0.3mmol/L DPPH ethanol solution was added, and after completion of the reaction in the dark, the absorbance value of the mixture was measured at a wavelength of 517 nm. VC was used as a control.

Wherein: a is that ₀ Absorbance value of blank control, A ₁ For the absorbance value of the sample solution, A ₂ Is the absorbance value of absolute ethanol and a sample solution. The results are shown in FIG. 1.

2) Hydroxyl radical clearance: preparing a hydroxyl free radical system, adding distilled water to prepare hirudin samples with mass concentrations of 10, 5, 2.5, 1.25 and 0.625mg/mL, sequentially adding a ferrous sulfate solution with mass concentration of 0.2mL of 6mmol/L and a salicylic acid-ethanol solution into the reaction system, adding hydrogen peroxide solutions with mass concentrations of 0.2mL and 0.2mL of 6mmol/L respectively into the sample solutions, reacting for 1h at 37 ℃, and measuring the absorbance value of the mixture at the wavelength of 510nm after the reaction is completed. VC was used as a control.

Wherein: a is that ₀ Absorbance value of blank control, A ₁ For the absorbance value of the sample, A ₂ Background absorption for samples without salicylic acid-ethanol solution. The results are shown in FIG. 2.

3) Superoxide radical scavenging rate: preparing a hydroxyl free radical system, adding a prepared hirudin sample solution and 4.7mL of 50mmol/L Tris-HCl buffer solution (pH 8.2), uniformly mixing, keeping the temperature in a water bath at 25 ℃ for 20min, taking out, immediately adding 0.3mL of 3mmol/L pyrogallol solution preheated at 25 ℃, rapidly uniformly mixing, measuring the absorbance at 325nm, measuring the absorbance for 1 time every 30s, continuously measuring for 5min, and calculating the slope delta Ai and delta A0 of the plot of the absorbance of the sample and blank control and the reaction time in a linear range. VC was used as a control group. The clearance rate is calculated according to the formula. The results are shown in FIG. 3.

Superoxide radical scavenging% = (1- Δa) _i /ΔA ₀ )*100

Wherein: Δai is the rate of autoxidation of the pyrogallol after addition of the hirudin solution; Δa0 is the rate of autoxidation of the pyrogallol.

3. Evaluation of anti-aging Activity in vivo

Grouping, modeling and administration of experimental animals

60 healthy Kunming mice are selected, the male and female mice are half, the weight is 18-22g, and the healthy Kunming mice are randomly divided into 6 groups, namely a normal group, a UVB model group, a positive control group, a hirudin high-dose group, a hirudin medium-dose group and a hirudin low-dose group, wherein 10 mice are bred in separate cages, and 5 mice are bred in each cage. The mice are bred by simulating the circadian law (8:00 on in the morning and 18:30 off in the evening), the ambient temperature is kept at 24-26 ℃, the relative humidity is 50-70%, all drinking water, feed and padding are sterilized by autoclaving, and the padding is replaced for 3 times every week. The molding is carried out by adopting the method of the related literature and improving. The mice were fed adaptively for one week and the backs of the mice were dehaired and prepared, and the model group (UVB group), positive control group, uvb+hirudin high dose group, uvb+hirudin medium dose group, uvb+hirudin low dose group, all mice were dehaired with depilatory on the backs of the skin, exposing the skin of about 3cm x 3cm size, once every 5 days. UVB irradiation was performed simultaneously on UVB group, uvb+hirudin (high, medium, low) group and positive control group, and the specific method is as follows: ultraviolet lamp (UVB) power 40W, light intensity was measured before irradiation. Before each irradiation, the lamp tube is preheated for 15min, the positions of the mouse boxes are rotated every 15min in the irradiation process, so that the consistency of the light intensity received by the mice in each cage is ensured, the irradiation time is 60min each time, and the light source is positioned at the position about 40cm above the mice, and the molding is continued for 30d. Normal mice were fed with normal light and daily back skin was smeared with the same volume of normal saline. The UVB group is coated with the same volume of physiological saline every day, the administration group is coated with the same volume of hirudin every day, the positive control group is coated with the same volume of vitamin E emulsion ointment, and after the UVB model group, the administration group and the positive control group wait for 60 minutes of absorption, the UVB model group, the administration group and the positive control group are simultaneously irradiated with ultraviolet rays for 60 minutes. The administration was continued for 30d. Drawing materials: on experiment day 31, after all mice had anesthetized, the back dehaired skin was isolated. Part of the dye is used for measuring skin moisture content, and part of the dye is used for preparing pathological sections and is used for HE staining and Masson staining; a portion of the skin was stored at-80℃for subsequent experiments. Skin moisture content was measured by desiccation: the skin of the mice is wiped by filter paper as much as possible, dehaired skin with the neck and back of about 1cm multiplied by 1cm is cut, the wet weight of the skin is accurately weighed, then the skin is put into an oven with the temperature of 80 ℃ for drying for 12 hours, after moisture is basically dispersed, the skin is taken out, and the dry weight of the skin is accurately weighed according to the following formula: skin moisture percentage (%) = (wet weight-dry weight)/wet weight x 100%, calculated.

1) Effect on moisture content of mouse skin: as shown in fig. 5, the skin moisture content was significantly reduced in the UVB group compared to the administration group, and the difference was statistically significant (P < 0.05).

2) Mouse skin tissue HE staining results: as shown in fig. 6, a normal group can be found, the skin structure is complete, smooth, and closely arranged; compared with the normal UVB group, the epidermis layer is abnormally thickened, the stratum corneum layer is seriously shed, and the dermis layer collagen fibers and elastic fibers are absent, broken and curled, loose in structure and disordered in arrangement. Compared with the UVB group, the epidermis of the UVB+hirudin group has no abnormal thickening, and the stratum corneum is orderly arranged relative to the complete dermis layer, so that disorder is avoided.

3) Results of Masson staining of mouse skin tissue: as shown in fig. 7, UVB group was found to blur the boundary between epidermis and dermis, significantly reduce the thickness of dermis, reduce collagen fiber content, and even generate voids; collagen fibers are unevenly distributed, the content is reduced, breakage occurs, and the arrangement is disordered. The thickness of the UVB+hirudin dermis is normal, collagen fibers are uniformly distributed, and the arrangement is compact and neat.

Experiments show that the Hirudo manillensis extract has the effects of scavenging DPPH free radical, and has strong capability of scavenging hydroxyl free radical and superoxide anion free radical, and has antioxidant effect; poecilobdella manillensis promotes proliferation and migration of skin fibroblasts and secretes collagen fibers to fill the wound surface, promotes wound surface contraction, accelerates wound surface healing, and has good treatment effect on ultraviolet injury of mouse skin.

4) The skin on the back was taken, excess fat and other connective tissue was removed, and its SOD, GSH-Px activity and MDA content were determined strictly according to the method provided by the kit. The results of the change in antioxidant enzyme activity in the skin of mice are shown in FIG. 8. Compared with the normal group, the activity of SOD and GSH-Px in the skin of a model group of mice after UVA irradiation is extremely obviously reduced, and the content of MDA is extremely obviously increased, which indicates that the UVA irradiation reduces the activity of SOD and GSH-Px in the skin, thereby reducing the scavenging capacity of ROS, increasing the content of lipid peroxidation products MDA and improving the oxidative stress level of the skin. Compared with the model group, the activity of SOD and GSH-Px in the skin of the mice in the high-dose group of hirudin is extremely obviously increased, which proves that the hirudin can inhibit the decrease of the activity of SOD and GSH-Px caused by UVA irradiation and reduce the peroxidation of cell lipid, thereby effectively reducing the skin oxidative stress level caused by UVA, reducing the damage degree of the ultraviolet damaged skin of the mice and having strong anti-radiation effect on the skin, thus the hirudin is applied to the preparation of medicaments or cosmetics for preventing and treating the ultraviolet damage.

Example 6

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 1

Example 7

Human safety evaluation test

The repeated open type smearing test uses forearm flexor side as the tested part, the area is 3×3cm2, and 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 10, 5mg (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 application test of table 2, and the results are recorded as in table 3.

TABLE 2 skin reaction evaluation criteria for skin repetitive open smear test

Table 3 results scores for each set

The evaluation criteria of the freeze-dried essence are shown in table 4;

table 4 evaluation criteria for lyophilized extracts

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
		Moisturizing/nourishing sensation	1-10 points, the higher the point, the more moist the skin feels in use

The evaluation results of the freeze-dried essence are shown in table 5;

table 5 evaluation results of lyophilized essence

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

Claims (4)

1. A freeze-dried essence of hirudin extract for resisting aging and ultraviolet injury is characterized by comprising freeze-dried powder and solvent liquid;

the freeze-dried powder comprises: hirudin extract, mannitol, oligopeptide-3, ceramide, trehalose and arginine;

the solvent liquid comprises: glycerol, propylene glycol, hydrolyzed sodium hyaluronate, dipotassium glycyrrhizinate, hexapeptide-9, butanediol, squalane, PEG castor oil, hydrolyzed collagen, nicotinamide and water;

the freeze-dried powder comprises, by mass, 1% -2% of hirudin extract, 4% -5% of mannitol, 31.5% -2% of oligopeptide, 0.5% -1% of ceramide, 0.8% -1% of trehalose, 0.08% -1% of arginine and the balance deionized water; the solvent liquid comprises the following components in percentage by mass: 8-10% of glycerol, 7-8% of propylene glycol, 1-2% of hydrolyzed sodium hyaluronate, 2-3% of dipotassium glycyrrhizinate, 91-2% of hexapeptide, 1-1.5% of butanediol, 8-9% of squalane, 7-8% of PEG castor oil, 1-2% of hydrolyzed collagen, 0.4-0.5% of nicotinamide and the balance of deionized water.

2. The freeze-dried essence of the hirudin extract with the functions of resisting aging and ultraviolet injury according to claim 1, wherein the freeze-dried powder comprises the following components in percentage by mass: 1% of hirudin extract, 3% of mannitol, 31% of oligopeptide, 0.5% of ceramide, 0.8% of trehalose, 0.05% of arginine and the balance of deionized water; the solvent liquid comprises the following components in percentage by mass: 6% of glycerin, 7% of propylene glycol, 0.8% of hydrolyzed sodium hyaluronate, 1% of dipotassium glycyrrhizinate, 90.5% of hexapeptide, 0.6% of butanediol, 5% of squalane, 5% of PEG castor oil, 0.5% of hydrolyzed collagen, 0.2% of nicotinamide and the balance of deionized water.

3. The freeze-dried essence of the hirudin extract with the functions of resisting aging and ultraviolet injury according to claim 1, wherein the freeze-dried powder comprises the following components in percentage by mass: 2% of hirudin extract, 5% of mannitol, 32% of oligopeptide-32%, 0.8% of ceramide, 1% of trehalose, 1% of arginine and the balance of deionized water; the solvent liquid comprises the following components in percentage by mass: 10% of glycerol, 8% of propylene glycol, 1% of hydrolyzed sodium hyaluronate, 2% of dipotassium glycyrrhizinate, 92% of hexapeptide, 1.5% of butanediol, 8% of squalane, 7% of PEG castor oil, 1% of hydrolyzed collagen, 0.4% of nicotinamide and the balance of deionized water.

4. The method for preparing the freeze-dried essence of the hirudin extract with the functions of resisting aging and ultraviolet injury according to claim 1, which is characterized by comprising the following steps of:

s1: dissolving hirudin in water to obtain solution, purifying, deodorizing, and filtering with 0.22 μm filter membrane to obtain hirudin extract;

s2: preparing a solution from raw materials such as hirudin extract, mannitol, oligopeptide-3, ceramide, trehalose, arginine, the balance deionized water and the like according to a proportion;

s3: shaking the solution uniformly, filtering with a 0.22 μm filter membrane to obtain solution A, standing for over 36h, and canning;

s4: vacuum freeze-drying the canned solution A to obtain freeze-dried powder;

s5: dissolving glycerol, propylene glycol, hydrolyzed sodium hyaluronate, dipotassium glycyrrhizinate, hexapeptide-9, butanediol, squalane, PEG castor oil, hydrolyzed collagen, nicotinamide and the balance deionized water in proportion, and sterilizing in an autoclave at constant temperature of 121 ℃ for 30min to obtain a solution B; standing for more than 24 hours, and canning;

s6: the ratio of the freeze-dried powder to the essence is 50mg:3mL of freeze-dried powder and solvent liquid are uniformly mixed to obtain the hirudin extract freeze-dried essence.

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