CN113509398A - Liposome freeze-dried powder with repairing and anti-aging effects and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a liposome freeze-dried powder with repairing and anti-aging effects and a preparation method thereof.

Description

Liposome freeze-dried powder with repairing and anti-aging effects and preparation method thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a liposome freeze-dried powder with repairing and anti-aging effects and a preparation method thereof.

Background

In recent years, the physiological functions of polypeptides in the living body have been receiving more and more attention. A large number of research results show that the bioactive polypeptide has small molecular weight and easy absorption, can be combined with a receptor in various ways, enhances the specificity to the receptor, enhances the permeability of the skin, has better stability, has good application prospect in the aspect of skin beauty, is more and more emphasized in the aspect of cosmetics, is added into the cosmetics to achieve the purposes of whitening and resisting aging, and is more and more widely applied to the cosmetics, such as anti-aging facial original milk, anti-aging facial masks and the like; cannabidiol (CBD), the major active ingredient of cannabinoids, is characterized by non-addictive properties, non-neurotoxic properties, etc. In recent years, scientists find that cannabidiol is a powerful antioxidant and has a series of physiological activity functions of blocking breast cancer metastasis, resisting epilepsy, resisting spasm, resisting rheumatoid arthritis, resisting anxiety, resisting insomnia and the like. In the aspect of skin external application, cannabidiol has the effects of resisting oxidation, delaying aging, resisting bacteria, diminishing inflammation, removing acne, resisting allergy and the like. However, cannabidiol is a fat-soluble compound and is insoluble in water, and for most cosmetics with high water content, the low solubility of cannabidiol severely limits the addition amount of cannabidiol, so that the ideal skin care effect cannot be achieved.

Although most of the existing cosmetic products containing polypeptide and cannabidiol can achieve the aim of repairing and protecting skin, the activity of the cosmetic products is greatly influenced by the environment, and meanwhile, due to the problems that the water solubility of CBD is poor, the light stability is poor, the stability of polypeptide raw materials is easily influenced by factors such as aqueous solution, temperature and the like, the existing cosmetic products of the category have short storage period and unstable skin-care effect. Therefore, in order to solve the instability of the CBD and the polypeptide components, the sterile lyophilized powder with multiple effects of repairing, removing wrinkles, diminishing inflammation and the like and high stability is developed, and is one of the key technologies for promoting the market development of CBD and peptide skin care products.

The patent CN110251466A discloses a cannabidiol liposome, which comprises phospholipid, cholesterol, cannabidiol and buffer salt solution, and the finally prepared liposome has high encapsulation efficiency, good water solubility and poor stability. The document patent CN110179687A discloses a multi-effect repairing polypeptide composition and an application thereof in cosmetics, wherein six polypeptides are compounded for the first time, so that the obtained cosmetics have comprehensive repairing effects of moisturizing, oxidation resistance, speckle fading, wrinkle resistance and sensitive irritation relieving, can enhance the glossiness and the compactness of skin, and can restore the health state of the skin, but have poor stability. The application of the liposome freeze-dried powder compounded by Cannabidiol (CBD) and polypeptide in the technical field of cosmetics is not found through search.

Disclosure of Invention

The invention aims to provide the liposome freeze-dried powder with the effects of resisting aging and repairing, further improves the effects of wrinkle lightening and anti-inflammatory repairing of raw materials by compounding polypeptide and Cannabidiol (CBD), simultaneously improves the water solubility and stability of the cannabidiol as the raw materials by a liposome coating process, and further improves the stability of liposome by combining the coating technology and the freeze-drying technology, thereby improving the stability of a coated active substance.

In order to achieve the purpose, the invention adopts the following technical scheme: a liposome freeze-dried powder with repairing and anti-aging effects comprises the following raw materials in parts by weight: 1-5 parts of cannabidiol liposome standby liquid, 1-5 parts of polypeptide liposome standby liquid, 20-30 parts of 20% mannitol injection, 0.01-0.15 part of serum albumin, 0.1-0.5 part of disodium hydrogen phosphate, 0.01-0.1 part of sodium dihydrogen phosphate, 0.1-1 part of trehalose, 0.1-1 part of glycine, 800.01-0.1 part of polysorbate-0.1 part of ultrapure water and 57-78 parts of ultrapure water.

Preferably, the feed comprises the following raw materials in parts by weight: 3 parts of cannabidiol liposome standby liquid, 2 parts of polypeptide liposome standby liquid, 25 parts of 20% mannitol injection, 0.05 part of serum albumin, 0.2 part of disodium hydrogen phosphate, 0.05 part of sodium dihydrogen phosphate, 0.5 part of trehalose, 0.5 part of glycine, 800.05 parts of polysorbate-and 68.7 parts of ultrapure water.

Preferably, the weight part ratio of the cannabidiol liposome standby liquid to the polypeptide liposome standby liquid is (3-5): (1-2).

Preferably, the preparation process of the cannabidiol liposome standby liquid comprises the following steps: accurately weighing cannabidiol, phospholipid and cholesterol, adding into an organic solvent, heating and stirring to fully dissolve the cannabidiol, the phospholipid and the cholesterol to obtain an organic phase; evaporating the organic phase on a rotary evaporator to remove the solvent, so that the residue forms a phospholipid film on the inner wall of the container; adding phosphate buffer solution into the suspension, and continuously performing rotary evaporation to enable the phospholipid film to fall off to obtain liposome suspension; and injecting the suspension into a high-pressure homogenizer for homogenizing for 2-3 times, filtering through a filter membrane of 100-200 nm after homogenizing, and extruding to obtain the cannabidiol liposome standby liquid.

Preferably, the preparation process of the polypeptide liposome standby liquid comprises the following steps: weighing polypeptide raw materials and a protein activity protective agent, and dissolving in phosphate buffer saline solution; weighing lecithin and sodium cholate, adding absolute ethyl alcohol, and performing ultrasonic dissolution in a water bath; carrying out rotary evaporation on an organic phase containing a lipid material at 35-45 ℃ to obtain a uniform and continuous lipid film, and fully removing residual organic solvent; cooling to normal temperature, adding phosphate buffer solution containing polypeptide, and rotating and shaking to make lipid film fully peel off container wall, and make lipid film fully hydrate to form uniform liposome suspension; and (3) carrying out ice-water bath ultrasonic treatment on the liposome suspension, and filtering the liposome suspension through a 0.22-micron microporous filter membrane to obtain a uniform polypeptide liposome standby solution.

Further, the protein activity protective agent is prepared from a composition of arginine hydrochloride and sucrose according to a mass ratio of 1: (3-5).

Further, the polypeptide raw material comprises one or more of oligopeptide 1, oligopeptide 3, conopeptide or collagen peptide.

Further, the weight part ratio of the lecithin to the sodium cholate is 1: (1.5-2).

In addition, the invention also provides a preparation method of the liposome freeze-dried powder with the efficacy of repairing and resisting aging, which comprises the following steps:

s1, selecting a sterilized container, adding 1/5 parts of ultrapure water, weighing disodium hydrogen phosphate, sodium dihydrogen phosphate, trehalose, glycine, serum albumin and polysorbate-80, sequentially adding into water, and stirring until completely dissolving for later use;

s2, adding the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid in the formula amount into the solution prepared in the step S1, stirring until the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid are completely dissolved, and filtering with a filter membrane of 0.22um to obtain filtrate for later use;

s3, adding the filtrate obtained in the step S2 into the remaining ultrapure water and 20% mannitol injection in formula amount, stirring uniformly, packaging and freeze-drying to obtain the sterile freeze-dried powder.

Further, the lyophilization process in the step S3 includes the following steps:

s31, pre-freezing: cooling to minus 40 +/-2 ℃ at the speed of 0.4-0.8 ℃/min, pre-freezing for 2-4h, and vacuumizing to reduce the pressure to 10-20 Pa;

s32, sublimation drying: heating to-25 ℃, keeping the temperature for 3-5 h, then gradually heating to-15 ℃ and 0 ℃, and keeping the temperature for 3-5 h and 1-2 h in sequence;

s33, analysis and drying: heating to 20-30 ℃, and keeping for 8-10 h.

Therefore, compared with the prior art, the invention has the following beneficial effects:

(1) the polypeptide and the cannabidiol are compounded for use, so that the effects of the prepared liposome freeze-dried powder on resisting aging, repairing skin, diminishing inflammation, removing red swelling and resisting wrinkles are effectively improved.

(2) The invention improves the water solubility of the cannabidiol raw material through the liposome encapsulation technology, avoids the problem of precipitation in aqueous products, improves the stability of active ingredients in the raw material through the combination of the liposome encapsulation technology and the freeze-drying technology, and ensures that the product can achieve the optimal effects of resisting senility, repairing skin, resisting wrinkles, diminishing inflammation and removing red swelling.

Drawings

FIG. 1 shows the repairing effect of the freeze-dried powders of examples 1 to 3 and the freeze-dried powders of comparative examples 3 to 4 on the proliferation promoting effect on epidermal cells, which are measured in an anti-wrinkle repairing function test.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

Example 1 Liposome lyophilized powder with repairing and anti-aging effects

The formula is as follows: 3g of cannabidiol liposome standby liquid, 2g of polypeptide liposome standby liquid, 25g of 20% mannitol injection, 0.1g of serum albumin, 0.2g of disodium hydrogen phosphate, 0.05g of sodium dihydrogen phosphate, 0.5g of trehalose, 0.5g of glycine, 800.05 g of polysorbate-and 68.5g of ultrapure water.

The preparation method of the cannabidiol liposome standby liquid comprises the following steps: accurately weighing 2g of cannabidiol, 50g of phospholipid and 8.4g of cholesterol, adding into absolute ethyl alcohol, heating and stirring to fully dissolve the cannabidiol, the phospholipid and the cholesterol to obtain an organic phase; evaporating the organic phase on a rotary evaporator to remove the solvent, so that the residue forms a phospholipid film on the inner wall of the container; adding phosphate buffer solution into the suspension, and continuously performing rotary evaporation to enable the phospholipid film to fall off to obtain liposome suspension; and injecting the suspension into a high-pressure homogenizer for homogenizing for 2 times, filtering through a 100nm filter membrane after homogenizing, and extruding to obtain the cannabidiol liposome standby liquid.

The preparation method of the polypeptide liposome standby liquid comprises the following steps: weighing 2g of oligopeptide 1, 1g of arginine hydrochloride and 3g of sucrose composition, and dissolving in phosphate buffered saline; weighing 4g of lecithin and 6g of sodium cholate, adding absolute ethyl alcohol, and performing ultrasonic dissolution in a water bath; performing rotary evaporation on the organic phase containing the lipid material at 40 ℃ to obtain a uniform and continuous lipid film, and sufficiently removing residual organic solvent; cooling to 25 deg.C, adding phosphate buffer solution containing polypeptide, and rotating and shaking to make lipid film sufficiently peel off container wall, and make lipid film sufficiently hydrate to form uniform liposome suspension; and (3) carrying out ice-water bath ultrasonic treatment on the liposome suspension, and filtering the liposome suspension through a 0.22-micron microporous filter membrane to obtain a uniform polypeptide liposome standby solution.

The preparation method of the liposome freeze-dried powder comprises the following steps:

s1, selecting a sterilized container, adding 1/5 parts of ultrapure water, weighing disodium hydrogen phosphate, sodium dihydrogen phosphate, trehalose, glycine, serum albumin and polysorbate-80, sequentially adding into water, and stirring until completely dissolving for later use;

s2, adding the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid in the formula amount into the solution prepared in the step S1, stirring until the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid are completely dissolved, and filtering with a filter membrane of 0.22um to obtain filtrate for later use;

s3, adding the filtrate obtained in the step S2 into the remaining ultrapure water and 20% mannitol injection in formula amount, stirring uniformly, packaging and freeze-drying to obtain the sterile freeze-dried powder.

The lyophilization process in step S3 specifically includes the following steps:

s31, pre-freezing: cooling to-40 + -2 deg.C at a rate of 0.6 deg.C/min, pre-freezing for 3 hr, and vacuumizing to reduce pressure to 15 Pa;

s32, sublimation drying: heating to-25 deg.C, maintaining for 4h, gradually heating to-15 deg.C, and maintaining for 4h and 1 h;

s33, analysis and drying: the temperature is raised to 30 ℃ and kept for 8 h.

Example 2 Liposome freeze-dried powder with repairing and anti-aging effects

The formula is as follows: 3g of cannabidiol liposome standby liquid, 1g of polypeptide liposome standby liquid, 20g of 20% mannitol injection, 0.01g of serum albumin, 0.1g of disodium hydrogen phosphate, 0.01g of sodium dihydrogen phosphate, 0.1g of trehalose, 0.1g of glycine, 800.01 g of polysorbate-and 79.07g of ultrapure water.

The preparation method of the cannabidiol liposome standby liquid comprises the following steps: accurately weighing 2g of cannabidiol, 50g of phospholipid and 8.4g of cholesterol, adding into absolute ethyl alcohol, heating and stirring to fully dissolve the cannabidiol, the phospholipid and the cholesterol to obtain an organic phase; evaporating the organic phase on a rotary evaporator to remove the solvent, so that the residue forms a phospholipid film on the inner wall of the container; adding phosphate buffer solution into the suspension, and continuously performing rotary evaporation to enable the phospholipid film to fall off to obtain liposome suspension; and injecting the suspension into a high-pressure homogenizer for homogenizing for 2 times, filtering through a 100nm filter membrane after homogenizing, and extruding to obtain the cannabidiol liposome standby liquid.

The preparation method of the polypeptide liposome standby liquid comprises the following steps: weighing 2g of oligopeptide 1, 1g of arginine hydrochloride and 3g of sucrose composition, and dissolving in phosphate buffered saline; weighing 4g of lecithin and 6g of sodium cholate, adding absolute ethyl alcohol, and performing ultrasonic dissolution in a water bath; performing rotary evaporation on the organic phase containing the lipid material at 40 ℃ to obtain a uniform and continuous lipid film, and sufficiently removing residual organic solvent; cooling to 25 deg.C, adding phosphate buffer solution containing polypeptide, and rotating and shaking to make lipid film sufficiently peel off container wall, and make lipid film sufficiently hydrate to form uniform liposome suspension; and (3) carrying out ice-water bath ultrasonic treatment on the liposome suspension, and filtering the liposome suspension through a 0.22-micron microporous filter membrane to obtain a uniform polypeptide liposome standby solution.

The preparation method of the liposome freeze-dried powder comprises the following steps:

s1, selecting a sterilized container, adding 1/5 parts of ultrapure water, weighing disodium hydrogen phosphate, sodium dihydrogen phosphate, trehalose, glycine, serum albumin and polysorbate-80, sequentially adding into water, and stirring until completely dissolving for later use;

s2, adding the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid in the formula amount into the solution prepared in the step S1, stirring until the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid are completely dissolved, and filtering with a filter membrane of 0.22um to obtain filtrate for later use;

s3, adding the filtrate obtained in the step S2 into the remaining ultrapure water and 20% mannitol injection in formula amount, stirring uniformly, packaging and freeze-drying to obtain the sterile freeze-dried powder.

The lyophilization process in step S3 specifically includes the following steps:

s31, pre-freezing: cooling to-40 + -2 deg.C at a rate of 0.4 deg.C/min, pre-freezing for 2 hr, and vacuumizing to reduce pressure to 10 Pa;

s32, sublimation drying: heating to-25 deg.C, maintaining for 3h, gradually heating to-15 deg.C, and maintaining for 3h and 1 h;

s33, analysis and drying: the temperature is raised to 30 ℃ and kept for 8 h.

Example 3 Liposome lyophilized powder with repairing and anti-aging effects

The formula is as follows: 5g of cannabidiol liposome standby liquid, 2g of polypeptide liposome standby liquid, 30g of 20% mannitol injection, 0.15g of serum albumin, 0.5g of disodium hydrogen phosphate, 0.1g of sodium dihydrogen phosphate, 1g of trehalose, 1g of glycine, 800.1 g of polysorbate-and 60.15g of ultrapure water.

The preparation method of the cannabidiol liposome standby liquid comprises the following steps: accurately weighing 2g of cannabidiol, 50g of phospholipid and 8.4g of cholesterol, adding into absolute ethyl alcohol, heating and stirring to fully dissolve the cannabidiol, the phospholipid and the cholesterol to obtain an organic phase; evaporating the organic phase on a rotary evaporator to remove the solvent, so that the residue forms a phospholipid film on the inner wall of the container; adding phosphate buffer solution into the suspension, and continuously performing rotary evaporation to enable the phospholipid film to fall off to obtain liposome suspension; and injecting the suspension into a high-pressure homogenizer for homogenizing for 2 times, filtering through a 100nm filter membrane after homogenizing, and extruding to obtain the cannabidiol liposome standby liquid.

The preparation method of the polypeptide liposome standby liquid comprises the following steps: weighing 2g of oligopeptide 1, 1g of arginine hydrochloride and 3g of sucrose composition, and dissolving in phosphate buffered saline; weighing 4g of lecithin and 6g of sodium cholate, adding absolute ethyl alcohol, and performing ultrasonic dissolution in a water bath; performing rotary evaporation on the organic phase containing the lipid material at 40 ℃ to obtain a uniform and continuous lipid film, and sufficiently removing residual organic solvent; cooling to 25 deg.C, adding phosphate buffer solution containing polypeptide, and rotating and shaking to make lipid film sufficiently peel off container wall, and make lipid film sufficiently hydrate to form uniform liposome suspension; and (3) carrying out ice-water bath ultrasonic treatment on the liposome suspension, and filtering the liposome suspension through a 0.22-micron microporous filter membrane to obtain a uniform polypeptide liposome standby solution.

The preparation method of the liposome freeze-dried powder comprises the following steps:

s1, selecting a sterilized container, adding 1/5 parts of ultrapure water, weighing disodium hydrogen phosphate, sodium dihydrogen phosphate, trehalose, glycine, serum albumin and polysorbate-80, sequentially adding into water, and stirring until completely dissolving for later use;

s2, adding the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid in the formula amount into the solution prepared in the step S1, stirring until the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid are completely dissolved, and filtering with a filter membrane of 0.22um to obtain filtrate for later use;

s3, adding the filtrate obtained in the step S2 into the remaining ultrapure water and 20% mannitol injection in formula amount, stirring uniformly, packaging and freeze-drying to obtain the sterile freeze-dried powder.

The lyophilization process in step S3 specifically includes the following steps:

s31, pre-freezing: cooling to-40 + -2 deg.C at a rate of 0.8 deg.C/min, pre-freezing for 4 hr, and vacuumizing to reduce pressure to 20 Pa;

s32, sublimation drying: heating to-25 deg.C, maintaining for 5h, gradually heating to-15 deg.C, and maintaining for 5h and 2 h;

s33, analysis and drying: the temperature is raised to 30 ℃ and kept for 8 h.

Comparative example 1

The comparative example differs from example 1 only in that: replacing the standby liquid phase of the cannabidiol liposome with cannabidiol, and replacing the standby liquid phase of the polypeptide liposome with a polypeptide raw material.

Preparation of lyophilized powder reference is made to example 1.

Comparative example 2

The formulation was the same as in example 1.

The preparation method is compared with example 1, and the comparative example only differs from example 1 in that: the solution prepared in step S3 was not subjected to lyophilization.

Comparative example 3

The comparative example differs from example 1 only in that: the CBD-free liposome stock solution is not contained, and the content of the polypeptide liposome stock solution is correspondingly increased to 5 g.

The preparation process is referred to example 1.

Comparative example 4

The comparative example differs from example 1 only in that: the stock solution of the polypeptide-free liposome is correspondingly increased to 5 g.

The preparation process is referred to example 1.

Experimental example I stability test

Firstly, experimental samples: freeze-dried powder samples of examples 1-3 and samples of comparative examples 1-2.

Second, Experimental methods

2.1 temperature stability test

Placing the freeze-dried powder obtained in the embodiment 1-3 and the sample obtained in the comparative example 1-2 in an environment of minus 15 +/-2 ℃ and 45 +/-2 ℃ for one month respectively, then dissolving the sample in distilled water according to the concentration of 100mg/mL, and observing whether the solution has the phenomenon of layering or precipitation.

2.2 Long-term storage stability test

The freeze-dried powder of the embodiment 1-3 and the sample of the comparative example 1-2 are placed at the room temperature of 25 +/-5 ℃ for 24 months, taken out at 0, 3, 6, 12, 18 and 24 months, dissolved in distilled water according to the concentration of 100mg/mL, observed whether the precipitation phenomenon exists or not, and recorded the re-dissolubility.

Third, experimental results

The experimental results are shown in tables 1-2.

TABLE 1 results of temperature stability test of each sample

As can be seen from Table 1, examples 1 to 3 of the present invention all maintain good stability at different temperatures, and are superior to comparative examples 1 to 2; the results of the examples 1-3 and the comparative examples 1-2 prove that the combination of the liposome encapsulation technology and the freeze-drying technology effectively improves the stability of the final product, and ensures that the prepared liposome freeze-dried powder has higher effects of diminishing inflammation, repairing, resisting aging, removing wrinkles and the like.

TABLE 2 Long-term storage stability test results

As can be seen from the data in Table 2, examples 1 to 3 of the invention still have good stability in a long-term storage experiment for 24 months, have strong re-solubility and are all superior to comparative examples 1 to 2; from the results of examples 1-3 and comparative examples 1-2, it can be known that the liposome encapsulation technology of the present invention can effectively improve the water solubility of cannabidiol as a raw material, and the combination of the liposome encapsulation technology and the freeze-drying technology further enhances the stability of the final product, while maintaining good re-solubility.

Experimental example II in vitro Oxidation resistance test

Firstly, experimental samples: freeze-dried powder of examples 1-3 and freeze-dried powder of comparative examples 3-4.

Second, Experimental methods

2.1DPPH clearance assay

Dissolving 0.5g of freeze-dried powder obtained in examples 1-3 and freeze-dried powder obtained in comparative examples 3-4 in an ethanol solution, diluting to 0.5mg/mL, putting 2mL of sample solution of 0.5mg/mL in a test tube, adding 2mL of DPPH solution (0.1 mmol/L prepared by absolute ethanol), mixing uniformly, reacting in the dark for 30min, and measuring the light absorption value at 517nm of a spectrophotometer. The blank experiment is carried out by replacing the sample solution with water, and the sample interference experiment is carried out by replacing the DPPH solution with absolute ethyl alcohol. The sample clearance for DPPH radicals was calculated and IC50 was calculated by linear fitting. The DPPH clearance calculation formula is shown in formula (1).

DPPH clearance (%) ([ 1- (a1-a/a0) ] × 100% (1)

In the formula: a0 is absorbance of blank; a1 is the absorbance of the sample set; a is the absorbance of the interfering group.

2.2 scavenging of hydroxyl radicals

2.2mmol/L H2O2, 2.25mmol/L FeSO4 aqueous solution, 2.25mmol/L salicylic acid 95% (volume fraction) ethanol solution 0.067mL each, and sample solution 0.1mL are added into a 96-well plate, and the mixture is reacted for 5min in the dark at room temperature, and then the absorbance is detected at 510 nm. The blank control group was prepared by repeating 3 wells for each concentration using 0.1mL of sample solvent in place of the sample solution, and the clearance was calculated as equation (2) and IC50 was calculated by linear fitting.

Hydroxyl radical clearance rate (A1-A2)/A1X 100% (2)

In the formula: a1 is the average absorbance of the blank; a2 is the average absorbance of the sample.

Third, experimental results

The results of the data obtained by the experiment are shown in tables 3-4.

TABLE 3 sample scavenging ability for DPPH free radical

TABLE 4 scavenging ability of the samples for hydroxyl radicals

As can be seen from the data in table 3, the liposome lyophilized powders prepared in embodiments 1 to 3 of the present invention all have strong DPPH free radical scavenging ability; the IC50 values of examples 1 to 3 are lower than those of comparative examples 3 to 4, and it can be seen that examples 1 to 3 of the present invention have higher oxidation resistance than comparative examples 3 to 4.

As can be seen from the data in Table 4, the freeze-dried powders obtained in the embodiments 1 to 3 of the present invention all have good hydroxyl radical scavenging ability; meanwhile, compared with the IC50 values of comparative examples 3 to 4, the examples 1 to 3 of the present invention have better oxidation resistance. The data in tables 3-4 prove that the antioxidant and anti-aging capacity of the freeze-dried powder can be effectively improved by the compound use of the polypeptide and the cannabidiol.

Experimental example III evaluation of skin irritation safety of product

Firstly, experimental samples: freeze-dried powder of examples 1-3, sample of comparative example 1 and physiological saline.

Second, Experimental methods

Adopting an autologous control mode, selecting 20 white rabbits with healthy, adult and undamaged skins, randomly dividing the rabbits into 5 groups of 4 rabbits, and adopting a homosomal left-right self-comparison method. Removing the back hair on two sides of the spine of the test animal by 8% sodium sulfide 24h before the test, removing the area of the back hair by about 3cm multiplied by 3cm, dissolving 0.5mL of samples of examples 1-3 and comparative example 1 by using a solvent into a solution, coating the solution on one side, using the other side as a blank control, coating once a day, cleaning after coating, observing the state of the skin, and continuously coating and observing for 14 days. The following day, shearing hairs before each application, and removing residual test substance with water. After the skin irritation test paper is smeared for 1h, the result is observed, the experimental result is scored according to the skin irritation response scoring standard in 2015 edition of 'cosmetic hygiene standards', the integral mean value of the animals to be dosed is calculated, and the skin irritation intensity is judged according to a skin irritation intensity scoring table.

Skin irritation response scoring criteria: no erythema score 0, mild erythema (barely visible) score 1, moderate erythema (clearly visible) score 2, severe erythema score 3, purplish red erythema to mild eschar score 4; no edema was scored at 0 point, mild edema (barely visible) at 1 point, moderate edema (markedly elevated) at 2 points, severe edema (1 mm of skin with clear contour) at 3 points, and severe edema (1 mm or more of skin with enlargement) at 4 points.

Skin irritation response score mean (total score for erythema and eschar + total score for edema)/total number of experimental animals, with the score for each group being the highest for different observation times. Skin irritation intensity grading criteria: score <0.5, no irritation; score 0.5 to <2.0, mild irritability; scoring for 2.0-6.0, and moderate irritation; score >6.0, strong irritation. The results of the experimental scoring are shown in table 3.

Third, experimental results

The results of the experimental scoring are shown in table 4.

Table 4 skin irritation response score results

As can be seen from the experimental data in Table 4, no irritation such as erythema and edema is generated on the skin of the experimental rabbit in the examples 1 to 3 and the normal saline group, and the average reaction irritation strength scores are all 0; comparative example 1 sample fluid exhibited a barely visible erythema 24h after application but returned to normal within 48h, with a mean score of 0.25<0.5 and no irritation.

Skin damage group comparative example 1, barely visible erythema edema occurred at 24h and all returned to normal within 48h, and no erythema or edema was observed in other groups except for the dark red scab that occurred after the blade scratched normal skin.

In conclusion, the freeze-dried powder of embodiments 1 to 3 of the present invention has no irritation to skin when used.

Experimental example four, anti-wrinkle repair function test

First, repair function test

1. Sample preparation: the freeze-dried powder obtained in the embodiment 1-3 and the freeze-dried powder obtained in the comparative example 3-4 are weighed in equal amount, respectively dissolved in 1mL of ultrapure water, filtered and sterilized by a 0.22 micron sterile filter, and prepared into a sample solution, and the sample solution is stored at-20 ℃ for later use.

2. The detection method comprises the following steps: immortalized epidermal HACAT cells were seeded at a seeding density of 6X 104cells/mL into 96-well cell culture plates and cultured overnight. The sample solutions of examples 1 to 3 and comparative examples 3 and 4 were diluted in 1640 cell culture medium (containing 0.4% fetal bovine serum) at the same fold ratio to 6 concentration gradients, the plate medium was discarded, and the sample dilutions were replaced with 100. mu.l/well and 4 duplicate wells at each concentration to set cell blanks. And (5) continuing to culture for 48 hours, and detecting the proliferation condition of the cells by using a CCK8 detection kit. Adding 10 microliters of CCK-8 reagent solution into each well, continuously culturing for 1-4 hours, uniformly mixing for 1 minute by using a low-speed shaking table, detecting the wavelength at 450nm, and detecting the OD value on an enzyme-labeling instrument.

Cell proliferation rate (%) < percent [ (experimental OD-blank OD value) - (cell control OD value-blank control) ]/(cell control OD value-blank control) × 100%

3. The result of the detection

The results are shown in FIG. 1.

Compared with the freeze-dried powder of comparative examples 3-4, the freeze-dried powder of examples 1-3 of the invention has higher cell proliferation rate under different concentrations, and the highest cell proliferation rate is 48% when the concentration of example 1 is 10mg/ml, so that experimental data can prove that the skin repair function of the freeze-dried powder can be effectively enhanced by the combined use of cannabidiol and polypeptide.

Second, anti-wrinkle effect detection

1. And (3) testing a sample: the freeze-dried powders prepared in the embodiments 1-3, the comparative example 3 and the comparative example 4 are tried by a subject, a sample is dissolved by purified water before use, and the storage form of the undissolved sample is normal temperature storage.

2. The test population: healthy women, age: skin condition between 35 and 45 years of age: the skin conditions are approximately similar, dry-neutral skin, the degree of facial wrinkles is not very different, and no allergic history of skin diseases exists. Each group contained 10 subjects.

3. Test method

The same sample was used for each group of subjects. The skin of these volunteers was not allergic to cosmetics, and the use of other cosmetics was prohibited during the experiment. Volunteers took eight points in the morning and nine points in the evening every day, cleaned the face, wiped dry facial water, taken a proper amount of sample and smeared on the whole face, and then gently massaged by finger and abdomen to promote the sample to be fully absorbed. The experimental period is one month, Skin elasticity of 5 fixed points on the face of the subject is detected by using a Skin detector of Skin-SP after one month, and the reduction degree of the total number of wrinkles on the face of the subject is detected by using a Skin detector MC-880.

4. Results of the experiment

The results of the experimental tests are shown in table 5.

Table 5 test results for lyophilized powder of example 1

As can be seen from table 5, compared with comparative examples 3 and 4, the liposome freeze-dried powder prepared in examples 1 to 3 of the present invention has significantly improved skin elasticity index and significantly reduced wrinkle amount, and the freeze-dried powder of the present invention has the effects of wrinkle removal, skin repair, and skin elasticity improvement.

From the experiments, the effects of the raw material components of the freeze-dried powder in the aspects of wrinkle removal, inflammation resistance and repair are improved by compounding the polypeptide raw material and the cannabidiol, and the water solubility and the stability of the raw material cannabidiol are further improved by combining a liposome coating technology and a freeze-drying technology.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (10)

1. The liposome freeze-dried powder with the effects of repairing and resisting aging is characterized by comprising the following raw materials in parts by weight: 1-5 parts of cannabidiol liposome standby liquid, 1-5 parts of polypeptide liposome standby liquid, 20-30 parts of 20% mannitol injection, 0.01-0.15 part of serum albumin, 0.1-0.5 part of disodium hydrogen phosphate, 0.01-0.1 part of sodium dihydrogen phosphate, 0.1-1 part of trehalose, 0.1-1 part of glycine, 800.01-0.1 part of polysorbate-0.1 part of ultrapure water and 57-78 parts of ultrapure water.

2. The lyophilized liposome powder of claim 1, comprising the following raw materials in parts by weight: 3 parts of cannabidiol liposome standby liquid, 2 parts of polypeptide liposome standby liquid, 25 parts of 20% mannitol injection, 0.05 part of serum albumin, 0.2 part of disodium hydrogen phosphate, 0.05 part of sodium dihydrogen phosphate, 0.5 part of trehalose, 0.5 part of glycine, 800.05 parts of polysorbate-and 68.7 parts of ultrapure water.

3. The freeze-dried liposome powder of claim 1 or 2, wherein the weight part ratio of the cannabidiol liposome stock solution to the polypeptide liposome stock solution is (3-5): (1-2).

4. The lyophilized liposome powder of claim 1 or 2, wherein the cannabidiol liposome stock solution is prepared by a process comprising: accurately weighing cannabidiol, phospholipid and cholesterol, adding into an organic solvent, heating and stirring to fully dissolve the cannabidiol, the phospholipid and the cholesterol to obtain an organic phase; evaporating the organic phase on a rotary evaporator to remove the solvent, so that the residue forms a phospholipid film on the inner wall of the container; adding phosphate buffer solution into the suspension, and continuously performing rotary evaporation to enable the phospholipid film to fall off to obtain liposome suspension; and injecting the suspension into a high-pressure homogenizer for homogenizing for 2-3 times, filtering through a filter membrane of 100-200 nm after homogenizing, and extruding to obtain the cannabidiol liposome standby liquid.

5. The lyophilized liposome powder of claim 1 or 2, wherein the preparation process of the polypeptide liposome solution for later use comprises: weighing polypeptide raw materials and a protein activity protective agent, and dissolving in phosphate buffer saline solution; weighing lecithin and sodium cholate, adding absolute ethyl alcohol, and performing ultrasonic dissolution in a water bath; carrying out rotary evaporation on an organic phase containing a lipid material at 35-45 ℃ to obtain a uniform and continuous lipid film, and fully removing residual organic solvent; cooling to normal temperature, adding phosphate buffer solution containing polypeptide, rotating and shaking at normal temperature to make lipid film fully peel off container wall, and making lipid film fully hydrate to form uniform liposome suspension; and (3) carrying out ice-water bath ultrasonic treatment on the liposome suspension, and filtering the liposome suspension through a 0.22-micron microporous filter membrane to obtain a uniform polypeptide liposome standby solution.

6. The lyophilized liposome powder of claim 5, wherein the polypeptide material comprises one or more of oligopeptide 1, oligopeptide 3, conopeptide or collagen peptide.

7. The lyophilized liposome powder of claim 5, wherein the protein activity protector is prepared from a composition of arginine hydrochloride and sucrose in a mass ratio of 1: (3-5).

8. The lyophilized liposome powder of claim 5, wherein the weight portion ratio of lecithin to sodium cholate is 1: (1.5-2).

9. The preparation method of the freeze-dried liposome powder with the effects of repairing and resisting aging as claimed in any one of claims 1 to 8, which comprises the following steps:

s1, selecting a sterilized container, adding 1/5 parts of ultrapure water, weighing disodium hydrogen phosphate, sodium dihydrogen phosphate, trehalose, glycine, serum albumin and polysorbate-80, sequentially adding into water, and stirring until completely dissolving for later use;

s2, adding the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid in the formula amount into the solution prepared in the step S1, stirring until the cannabidiol liposome standby liquid and the polypeptide liposome standby liquid are completely dissolved, and filtering with a filter membrane of 0.22um to obtain filtrate for later use;

s3, adding the filtrate obtained in the step S2 into the remaining ultrapure water and 20% mannitol injection in formula amount, stirring uniformly, packaging and freeze-drying to obtain the sterile freeze-dried powder.

10. The method for preparing liposome lyophilized powder with repairing and anti-aging effects as claimed in claim 9, wherein the lyophilization process in step S3 comprises the following steps:

s31, pre-freezing: cooling to minus 40 +/-2 ℃ at the speed of 0.4-0.8 ℃/min, pre-freezing for 2-4h, and vacuumizing to reduce the pressure to 10-20 Pa;

s32, sublimation drying: heating to-25 ℃, keeping the temperature for 3-5 h, then gradually heating to-15 ℃ and 0 ℃, and keeping the temperature for 3-5 h and 1-2 h in sequence;

s33, analysis and drying: heating to 20-30 ℃, and keeping for 8-10 h.

Images