JP7186268B2 - Schizophyllan liposome and its preparation method and use - Google Patents

Description

The present invention belongs to the technical field of cosmetics, and relates to schizophyllan liposomes and preparation methods and uses thereof, particularly schizophyllan liposomes with small particle size, uniform distribution and high encapsulation efficiency, and preparation methods and uses thereof. Regarding.

Schizophyllan is a water-soluble polysaccharide extracted from the fruiting body, mycelium, or fermented liquid of Suehirotake. In recent years, researches on schizophyllans are mainly concentrated in the fields of biological properties, food and medicinal value, acclimatization and deep fermentation of S. japonicus.

Schizophyllan also has moisturizing and anti-aging effects and can be added to cosmetics as an active ingredient, and there are reports in the prior art on the use of schizophyllan in cosmetics. For example, CN107007525A discloses a composition with long-lasting and effective moisturizing function, which comprises schizophyllan, Dendrobium nobile extract, Boulevard extract, Dianthus japonicum extract, sodium pyrrolidone carboxylate, hyaluronic acid Comprising sodium, ceramide, polyol and water, the composition is used to prepare moisturizing cosmetics, and has the advantages of good immediate moisturizing effect and long-lasting moisturizing effect. For example, CN107595724A discloses a skin care matrix comprising deionized water, modified schizophyllan, 1,3-butanediol, phenoxyethanol, durvillea antarctica extract and chicory root extract. Containing, the skin care matrix can alleviate the technical problem that skin care products in the prior art have only one function, and at the same time have the effects of anti-aging, moisturizing and repairing.

However, when schizophyllan is added to cosmetics as a high-molecular-weight, water-soluble natural product, it cannot penetrate and be retained in the skin in a free state, limiting its moisturizing or anti-aging effects.

In view of the shortcomings of the prior art, the object of the present invention is to provide schizophyllan liposomes and preparation methods and uses thereof, especially schizophyllan liposomes with small particle size, uniform distribution and high encapsulation efficiency, and preparation methods thereof. to provide use.

In order to achieve the object of the present invention, the present invention has taken the following technical solutions.
In a first aspect, the present invention provides a method for preparing schizophyllan liposomes, said method comprising:
a step (1) of dissolving a lipid in an organic solvent and removing the organic solvent to form a lipid membrane;
The lipid film obtained in step (1) is mixed with schizophyllan, a surfactant and a buffer solution and hydrated until the lipid film separates and forms a milky liquid to obtain a crude suspension of schizophyllan liposomes. step (2);
and step (3) of sonicating the crude suspension of schizophyllan liposomes obtained in step (2) and filtering through a filtration membrane to obtain said schizophyllan liposomes.

Schizophyllan, as a high-molecular-weight, water-soluble natural product, cannot penetrate and be retained in the skin in a free state when added to cosmetics, limiting its moisturizing or anti-aging effects. In order to overcome this shortcoming, the present invention encapsulates schizophyllan in liposomes to prepare a schizophyllan liposome product that penetrates the skin and is easier to absorb and retain in the skin. Significantly improve the utilization rate and exhibit better moisturizing and anti-aging effects. However, it is not easy to encapsulate schizophyllan in liposomes, it is difficult to control the particle size and dispersibility of liposomes, and it is difficult to improve encapsulation efficiency. It was found that a schizophyllan liposomal product with small particle size, uniform dispersion and high encapsulation efficiency can be obtained, and has good moisturizing, whitening and antioxidant effects.

Preferably, the lipid according to step (1) comprises lecithin and cholesterol.

Preferably, the mass ratio of said lecithin and cholesterol is 1:(0.05-0.35), such as 1:0.05, 1:0.10, 1:0.15, 1:0.20. , 1:0.25, 1:0.30 or 1:0.35, etc., and other specific point values within the above numerical ranges are all selectable and will not be described here.

In the preparation method of the present invention, the lipids are specifically selected from lecithin and cholesterol and mixed in a specific mixing ratio relationship, so that the encapsulation efficiency and drug loading of the product prepared by this combination method are improved. higher, its percutaneous absorption and retention in the skin are higher, and it has better moisturizing, whitening and anti-aging effects.

Preferably, the organic solvent according to step (1) comprises any one or a combination of at least two of methanol, ethanol, ethyl ether, acetone or dichloromethane, preferably a combination of ethyl ether and methanol.

The combination of at least two types is, for example, a combination of ethanol and ethyl ether, a combination of methanol and ethanol, a combination of acetone and dichloromethane, etc. Any other combination method can be selected, and the description is omitted here. do.

Preferably, the volume ratio of said ethyl ether to methanol is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, the above values All other specific point values within the range are selectable and will not be described here.

Preferably, removing the organic solvent according to step (1) uses a rotary evaporation method.

Preferably, the surfactant according to step (2) is polysorbate 80, polyglyceryl-10 myristate, polyglyceryl-6 caprylate, PEG-20 methylglucose sesquistearate, PEG-10 dimethicone, ceteareth-6, methylgluceth- 20, glyceryl stearate, cetearyl oleate or sorbitan oleate, or a combination of at least two of them, preferably a combination of polysorbate 80 and polyglyceryl-10 myristate.

Preferably, the weight ratio of said polysorbate 80 to polyglyceryl-10 myristate is (1.5-5):1, such as 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 5:1, etc., and other specific point values within the above numerical ranges are all selectable and will not be described here.

In the preparation method according to the present invention, the surfactant is specifically selected from the combination of polysorbate 80 and polyglyceryl-10 myristate, and is blended in a specific blending ratio relationship, so the product prepared by this combination method The particle size distribution is more uniform, the encapsulation efficiency and drug loading are higher, the percutaneous absorption and skin retention are higher, and the moisturizing, whitening and anti-aging effects are better.

Preferably, said buffer comprises a phosphate buffer and has a pH of 6.5-7.5, eg pH=6.5, pH=6.7, pH=6.8, pH=7. 0, pH=7.2, pH=7.4 or pH=7.5, etc., and other specific point values within the above numerical ranges are all selectable and will not be described here.

Preferably, the schizophyllan according to step (2) refers to a schizophyllan stock solution, and the schizophyllan stock solution is a β-glucan solution with a mass concentration of 0.5% to 5% obtained by fermentation of Suehirotake.

Preferably, the mass ratio of said schizophyllan stock solution to lecithin is (0.01-0.25):1, such as 0.01:1, 0.05:1, 0.08:1, 0.10: 1, 0.12:1, 0.15:1, 0.20:1 or 0.25:1, etc., and all other specific point values within the above numerical ranges are selectable and are hereby incorporated by reference. We omit the explanation here.

Preferably, the mass ratio of said schizophyllan stock solution to surfactant is (2-10):1, such as 2:1, 3:1, 4:1, 5:1, 8:1 or 10:1. , and all other specific point values within the above numerical range are selectable and will not be described here.

Preferably, the mass ratio of said schizophyllan stock solution to buffer solution is 1:(5-20), such as 1:5, 1:8, 1:10, 1:12, 1:15, 1:16, 1 :18 or 1:20, etc., and other specific point values within the above numerical range are all selectable and will not be described here.

The schizophyllan undiluted solution, lecithin, surfactant, and buffer meet a specific mass ratio relationship, so that the prepared liposomes have a more uniform and stable particle size distribution, and the encapsulation efficiency and drug loading are improved. higher, its percutaneous absorption and retention in the skin are higher, and it has better moisturizing, whitening and anti-aging effects.

Preferably, the ultrasonic power of the ultrasonic treatment according to step (3) is between 100 and 600 W, such as 100 W, 200 W, 300 W, 400 W, 500 W or 600 W, other specifics within the above numerical ranges. Any point value can be selected, and the description is omitted here.

Preferably, the sonication time of the sonication according to step (3) is 20-40 min, such as 20 min, 25 min, 30 min, 35 min or 40 min, other specific values within the above numerical ranges. All point values are selectable, and the description is omitted here.

Preferably, the filtration membrane according to step (3) is a 0.45 μm micropore filtration membrane.

Preferably, the particle size of the schizophyllan liposomes obtained according to step (3) is between 50 and 200 nm, such as 50 nm, 60 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm or 200 nm, and the above numerical ranges All other specific point values in are selectable and will not be described here.

As a preferred technical solution of the present invention, the method for preparing the schizophyllan liposome is specifically:
Step (1) of dissolving lecithin and cholesterol in a mixed solvent of ethyl ether and methanol at a mass ratio of 1:(0.05 to 0.35), removing the organic solvent, and forming a lipid membrane;
The lipid film obtained in step (1) is mixed with a schizophyllan stock solution, a surfactant, and a phosphate buffer, and hydrated until the lipid film separates to form a milky liquid, yielding a crude suspension of schizophyllan liposomes. A step of obtaining a solution, wherein the mass ratio of schizophyllan stock solution and lecithin is (0.01-0.25): 1, the mass ratio of schizophyllan stock solution and surfactant is (2-10): 1, schizophyllan stock solution and buffer A step (2) in which the mass ratio of the liquid is 1: (5 to 20);
The crude suspension of schizophyllan liposomes obtained in step (2) is sonicated at 100-600 W for 20-40 minutes and filtered through a 0.45 μm micropore filtration membrane to obtain schizophyllan liposomes with a particle size of 50-200 nm. and step (3) of obtaining

In a second aspect, the present invention provides schizophyllan liposomes prepared by the method for preparing schizophyllan liposomes according to the first aspect.

In a third aspect, the invention provides the use of schizophyllan liposomes according to the second aspect in the preparation of a cosmetic product.

Compared with the prior art, the present invention has the following beneficial effects.
Schizophyllan, as a high-molecular-weight, water-soluble natural product, cannot penetrate and be retained in the skin in a free state when added to cosmetics, limiting its moisturizing or anti-aging effects. In order to overcome this shortcoming, the present invention encapsulates schizophyllan in liposomes to prepare a schizophyllan liposome product that penetrates the skin and is easier to absorb and retain in the skin. Significantly improve the utilization rate and exhibit better moisturizing and anti-aging effects. The preparation method according to the present invention can obtain schizophyllan liposomal products with small particle size (115.97 nm to 466.38 nm), uniform dispersion, and high encapsulation efficiency (80% or more), and good It has been found to have moisturizing, whitening, and antioxidant effects.

FIG. 1 is an optical microscopic view of schizophyllan liposomes according to the present invention.

Hereinafter, the technical solution of the present invention will be further described through specific embodiments. It should be understood by those skilled in the art that the above examples are merely for the understanding of the invention and should not be viewed as specifically limiting the invention.

The schizophyllan stock solution according to the following examples was purchased from Guangzhou Qingtian Biotechnology Co., Ltd., model number was schizophyllan-G1.

Example 1
This example provides schizophyllan liposomes, the method of preparation of which was as follows.
(1) Lecithin and cholesterol were dissolved in a mixed solvent of ethyl ether/methanol with a mass ratio of 1:0.25 and a volume ratio of 2:1, and the organic solvent was removed by constant temperature water bath at 40°C and rotary evaporation under reduced pressure. , forming a uniform lipid membrane,
(2) The lipid membrane obtained in step (1) was treated with schizophyllan undiluted solution, a surfactant (a combination of polysorbate 80 and polyglyceryl-10 myristate at a mass ratio of 4:1), and a phosphate buffer of pH = 7.2. and hydrated until the lipid film separates to form a milky white liquid to obtain a crude suspension of schizophyllan liposomes, the mass ratio of schizophyllan stock solution to lecithin is 0.2:1, schizophyllan stock solution to The mass ratio of the surfactant is 6:1, the mass ratio of the schizophyllan stock solution and the buffer solution is 1:15,
(3) The crude suspension of schizophyllan liposomes obtained in step (2) is sonicated at 400 W for 30 minutes and filtered through a 0.45 μm micropore filtration membrane to remove said schizophyllan liposomes with a particle size of 50-200 nm. Obtained.

Example 2
This example provides schizophyllan liposomes, the method of preparation of which was as follows.
(1) dissolving lecithin and cholesterol in dichloromethane at a mass ratio of 1:0.1, bathing in constant temperature water at 40° C., rotary evaporation under reduced pressure to remove the organic solvent, and forming a uniform lipid film;
(2) The lipid membrane obtained in step (1) was treated with schizophyllan undiluted solution, a surfactant (a combination of polysorbate 80 and polyglyceryl-10 myristate at a mass ratio of 5:1), and a phosphate buffer of pH = 7.2. and hydrated until the lipid film separates to form a milky white liquid to obtain a crude suspension of schizophyllan liposomes, the mass ratio of schizophyllan stock solution to lecithin is 0.05:1, schizophyllan stock solution to The mass ratio of the surfactant is 10:1, the mass ratio of the schizophyllan stock solution and the buffer solution is 1:10,
(3) The crude suspension of schizophyllan liposomes obtained in step (2) is sonicated at 200 W for 40 minutes and filtered through a 0.45 μm micropore filtration membrane to remove said schizophyllan liposomes with a particle size of 50-200 nm. Obtained.

Example 3
This example provides schizophyllan liposomes, the method of preparation of which was as follows.
(1) Dissolve lecithin and cholesterol in a mixed solvent of ethyl ether/ethanol with a mass ratio of 1:0.35 and a volume ratio of 1:1, bathe in constant temperature water at 40°C, and remove the organic solvent by rotary evaporation under reduced pressure. , forming a uniform lipid membrane,
(2) The lipid membrane obtained in step (1) was mixed with schizophyllan undiluted solution, a surfactant (combination of polysorbate 80 and glyceryl stearate at a mass ratio of 3:1), and a phosphate buffer solution of pH = 7.2. Mix and hydrate until the lipid film separates to form a milky white liquid to obtain a crude suspension of schizophyllan liposomes, the mass ratio of schizophyllan stock solution to lecithin is 0.25:1, schizophyllan stock solution and surfactant The mass ratio of the agent is 5:1, the mass ratio of the schizophyllan stock solution and the buffer solution is 1:20,
(3) The crude suspension of schizophyllan liposomes obtained in step (2) is sonicated at 600 W for 20 minutes and filtered through a 0.45 μm micropore filtration membrane to remove said schizophyllan liposomes with a particle size of 50-200 nm. Obtained.

Reference example 1
This example provides schizophyllan liposomes, the only difference between the preparation method and Example 1 is that lecithin is replaced with an equal amount of soybean phospholipids, all other conditions remain the same, and the preparation method is also carried out. Consistent with Example 1.

Reference example 2
This example provides schizophyllan liposomes, the only difference between the preparation method and Example 1 is that the mass ratio of lecithin and cholesterol is 1:0.02, but the total amount of lipids is left unchanged. . All other conditions remained the same, and the preparation method was also consistent with Example 1.

Reference example 3
This example provides schizophyllan liposomes, the method of preparation of which differs from Example 1 only in that the mass ratio of lecithin and cholesterol is 1:0.45, but the total amount of lipids is left unchanged. . All other conditions remained the same, and the preparation method was also consistent with Example 1.

Reference example 4
This example provides schizophyllan liposomes, the method of preparation of which differs from Example 1 only by replacing the surfactant with an equal amount of ceteareth-6. All other conditions remained the same, and the preparation method was also consistent with Example 1.

Reference example 5
This example provides schizophyllan liposomes, the method of preparation of which differed from Example 1 only by replacing the surfactant with an equivalent amount of PEG-10 dimethicone. All other conditions remained the same, and the preparation method was also consistent with Example 1.

Example 9
This example provides schizophyllan liposomes, and the difference between the preparation method and Example 1 is that the amount of schizophyllan stock solution added is unchanged and the amount of surfactant added is changed. was only to make the ratio of 1:1. All other conditions remained the same, and the preparation method was also consistent with Example 1.

Example 10
This example provides schizophyllan liposomes, and the difference between the preparation method and Example 1 is that the amount of schizophyllan stock solution added is unchanged and the amount of surfactant added is changed. was only to make the ratio of 15:1. All other conditions remained the same, and the preparation method was also consistent with Example 1.

Comparative example 1
This comparative example provides schizophyllan liposomes, and the only difference between the preparation method and Example 1 is that no surfactant is added in step (2), all other conditions remain the same, and the preparation method is also Consistent with Example 1.

Evaluation test:
(1) Observation of morphology and analysis of particle size The liposomes prepared in Examples 1 to 10 and Comparative Example 1 were diluted 10-fold with distilled water, ultrasonically dispersed, and subjected to laser particle size analysis using a Mastersizer 2000 manufactured by Malvern Instruments, UK. The average particle size and distribution of the liposomes were analyzed using an analyzer, each sample was measured three times, the average value was taken, and the results are shown in Table 1.

The data in Table 1 show that the schizophyllan liposomal product according to the present invention has a small particle size that can be controlled from 115.97 nm to 466.38 nm.

The liposomes prepared in Example 1 were diluted 10-fold with distilled water and observed under a 400-fold optical microscope. As shown in FIG. It was relatively uniform, free from aggregation, and had good dispersibility.

(2) Accurately weigh 0.5 mL of the liposome suspension, dilute it 3-fold with PBS, add it to the column, use a Sephadex G-50 gel column, and elute with PBS. The flow rate was controlled at 1 mL/min, free schizophyllan was collected and hydrolyzed, and the content of schizophyllan was determined using the phenol-sulfuric acid method. In addition, 0.5 mL of the liposome suspension was taken, added to a mixed solvent of ethyl ether/methanol at a volume ratio of 2:1 to dissolve, schizophyllan was extracted, and the total amount of schizophyllan was measured by the above method. Capsule efficiency and drug loading were calculated by the following equations.

式中、ｍ_総はリポソーム懸濁液におけるシゾフィランの総質量を表し、ｍ_遊はリポソーム懸濁液における遊離シゾフィランの質量を表し、ｍ_脂質はリポソーム懸濁液における脂質の総量を表した。

where m _total represents the total mass of schizophyllan in the liposome suspension, m _free represents the mass of free schizophyllan in the liposome suspension, and m _lipid represents the total amount of lipid in the liposome suspension.

Table 2 shows the results.

The data in Table 2 show that the encapsulation efficiency and drug loading of the schizophyllan liposomal product according to the present invention are high and can reach up to 87.69% and 42.13%.

(3) Evaluation of percutaneous absorption effect In this experiment, the back skin of freshly slaughtered pigs was selected and studied. First, do the following to the skin of the pig, first remove the hair, avoid damaging the skin pores as much as possible when removing the hair, then treat the subcutaneous fat, first gently remove the excess fat using a scalpel. Scrape off, try not to damage the dermal layer of the skin as much as possible, wipe off the remaining fat repeatedly with isopropanol, finally wash repeatedly with physiological saline, place in a refrigerator at -20 ° C, thaw within 1 month, and PBS. and dried between filter papers before percutaneous experiments.

Drug percutaneous studies on isolated porcine skin were performed using the vertical Franz diffusion tank method. The skin was fixed between the supply chamber and the reception tank, the stratum corneum of the skin faced the supply chamber, the dermis layer faced the reception tank, the effective diffusion area of the diffusion tank was 1 ^cm2 , and the volume of the reception tank was It was 10 mL. The diffusion tank was placed in a constant temperature water bath at 36° C., 1 mL of the schizophyllan liposome suspension was added to the drug supply tank, a rotor was placed in the diffusion tank, and magnetic stirring was performed at 20 r/min. After the sample was added and allowed to balance for 10 minutes, timing was initiated to dispense 1 mL from the diffusion tank at 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 18, 24 hours, respectively. Sampled and measured, 1 mL of isothermal PBS was added. Measure the content of schizophyllan in the receiving liquid sampled at different times as described above, and calculate the cumulative permeation amount Qn (mg/cm ² ) per unit area of pig skin at each corresponding time point by the following formula, Samples in each group were tested in triplicate and averaged.

ただし、Ｃ_ｎ、Ｃ_ｉはｎ番目とｉ番目のサンプリングポイントで測定したシゾフィラン質量濃度（ｍｇ／ｍＬ）、Ｖ_ｎは受容タンクの総体積（ｍＬ）、Ｖ_０はサンプリング体積（ｍＬ）、Ａは拡散タンク上の豚皮の有効面積（ｃｍ^２）であった。

where C _n , C _i are schizophyllan mass concentrations (mg/mL) measured at the n-th and i-th sampling points, V _n is the total volume of the receiving tank (mL), V ₀ is the sampling volume (mL), A was the effective area (cm ² ) of the pig skin on the diffusion tank.

After 24 hours, the skin was removed, washed with ultrapure water to remove residual liquid from the sample, cut into pieces, transferred to a tissue homogenizer, thoroughly ground to homogenize, added an appropriate amount of ethanol, and centrifuged. , sonicated for 20 minutes, centrifuged at 5000 r/min for 10 minutes, collected the precipitate, redissolved in deionized water, measured the content of schizophyllan by the phenol-sulfuric acid method, and measured the content of schizophyllan in the skin. Retention was calculated.

Table 3 shows the results.

From the data in Table 3, the cumulative permeation amount in 24h skin and the retention amount in skin of the schizophyllan liposomal product according to the present invention are all significantly higher than that of schizophyllan, reaching a maximum of 45.16 mg/ ^cm2 and 363.16 mg/ ^cm2. I found that it can be done. Moreover, the composition and proportion of lipids in the liposomes and the composition and proportion of surfactants significantly affected the cumulative permeation and retention of liposomes in the skin.

Usage example 1
A skin care emulsion containing schizophyllan liposomes and a skin care emulsion containing schizophyllan liposomes prepared in Examples 1 to 3, 9, 10, Reference Examples 1 to 5 and Comparative Example 1 were prepared separately (the effective amount of schizophyllan in the emulsion is 1 %), tested in humans by volunteers. The preparation method of the skin care emulsion was as follows.

The compounding ratio of the raw materials was as follows.

(1) Each raw material of phase A was precisely weighed, placed in a beaker, heated to 85° C., and stirred for 15 minutes while being kept warm.
(2) Into another beaker, the ingredients of phase B (except methylparaben) were sequentially added. After heating to 70°C, methylparaben was added.
(3) Start stirring, slowly pour phase A into phase B, mix, then turn on the homogenizer and homogenize for 5 minutes at a speed of 3000 rpm.
(4) The mixed sample was stirred at 400 rpm and cooled to 45° C., each material of phase C and schizophyllan liposome or schizophyllan were added and stirred for 15 minutes until uniform to obtain a skin care emulsion.

Evaluation test:
The prepared skin care emulsion was evaluated from three aspects of moisturizing, whitening and anti-aging.

(1) About the moisturizing effect test Preparation before the test: (1) From 3 days before the test, do not use any product (cosmetics or topical medicines) on the test site, and before the experiment, the subject will hold the inside of both forearms together. I had to wash. (2) The measurement area should be marked on the inside of both forearms of the subject, the area of the experimental area is at least 3 cm x 3 cm, multiple areas can be marked simultaneously on the same arm, and each test area The spacing between was at least 1 cm. (3) should have sat quietly without water or drink for at least 20 minutes in a qualifying room before formal testing;

Test process: (1) The product application area and blank comparison area should be randomly distributed in defined areas on the left and right arms so that the positions of all products and areas are statistically balanced. . (2) The test sample was applied once at a dosage of (2.0±0.1) mg/cm ² . (3) Using a capacitance skin moisture meter, measure the moisture content of the skin in the application area and the comparison area of the product before use, 30 minutes after use, 60 minutes after use, and 120 minutes after use, respectively. After that, the moisture content (%) in the skin was tested after 180 minutes of use, and each area was measured at least three times in parallel, and then the average value was taken. (4) If the skin of the volunteer had an adverse reaction during the use of the product, the test was stopped immediately, the volunteer was treated appropriately, and the adverse reaction that appeared was recorded.

Table 4 shows the results.

From the data in Table 4, it was found that when the schizophyllan according to the present invention was applied to the preparation of a skin care emulsion, the resulting product had a remarkable effect on moisturizing the skin. And the composition and proportion of lipids in liposomes and the composition and proportion of surfactants all significantly affected the above effects of liposomes.

(2) Test of Whitening Effect The test process was as follows.
1. Test samples and controls Samples: products prepared in Examples 1-3, 9, 10, products prepared in Reference Examples 1-5, products prepared in Comparative Example 1, schizophyllan undiluted solution.
SELECTION CRITERIA: 40 adult Chinese women aged 20-45 years with facial skin problems such as mottling, dull skin, yellowish dullness, and uneven skin tone. We excluded those who had acute inflammation or other skin diseases, those who had a history of allergies or cosmetic allergies, and those who were pregnant or lactating.
2. Test Samples and Subjects VISIA-CR Facial Skin Imaging Tester Skin image analysis software FrameScan.
3. Test environment Constant temperature and humidity environment, temperature: 22°C ± 1°C, humidity: 50% ± 5%. Prior to testing, subjects were required to sit quietly in this environment for 20 minutes.
4, Test method Using VISIA-CR, before using the product, after 1 week, 2 weeks, and 1 month after using the product, take an image of the entire face of the subject, and use the software FrameScan to measure the brightness of the skin color cieL *. Change values were analyzed. Table 5 shows the results.

From the data in Table 5, it can be seen that when the schizophyllan according to the present invention is applied in the preparation of skin care emulsion, the resulting product has a remarkable effect on skin whitening. And the composition and proportion of lipids in liposomes and the composition and proportion of surfactants all significantly affected the above effects of liposomes.

(3) Antiaging Effect Test The test process was as follows.
1. Test samples and subject Samples: Examples 1-3, 9, 10, Reference Examples 1-5 , Comparative Example 1, Schizophyllan undiluted solution.
Inclusion criteria: 40 healthy women aged 40-50 years with acute facial inflammation or other skin disease, allergic predisposition or history of cosmetic allergy, pregnant or breastfeeding. excluded.
2. Test device VISIA-CR facial skin image tester 3. Test environment Constant temperature and humidity environment, temperature: 22°C ± 1°C, humidity: 50% ± 5%. Prior to testing, subjects were required to sit quietly in this environment for 20 minutes.
4. Test method After washing the face with warm water every morning and every night, the test sample was used around the eyes. Photographs were taken, and the area of wrinkles in the outer corner of the eye, the number of wrinkles, and the amount of change in depth of wrinkles were evaluated. Table 6 shows the test results.

From the data in Table 6, it was found that when the schizophyllan according to the present invention was applied to the preparation of a skin care emulsion, the resulting product had a remarkable anti-aging effect on the skin. And the composition and proportion of lipids in liposomes and the composition and proportion of surfactants all significantly affected the above effects of liposomes.

Although the present invention has illustrated the schizophyllan liposomes of the present invention and the preparation method and use thereof by the above examples, the present invention is not limited to the above examples, i.e., the present invention must be practiced by the above examples. Applicant states that it does not. For those skilled in the art, any modification to the present invention, equivalent substitution and addition of auxiliary ingredients to each raw material of the product of the present invention, selection of specific embodiments, etc., will all fall within the protection scope and disclosure scope of the present invention. Clear.

Although the preferred embodiments of the invention have been described in detail above, the invention is not limited to the specific details of the above embodiments, and various simple modifications can be made within the spirit of the invention. and all these simple modifications fall within the protection scope of the present invention.

It should be noted that each of the specific technical features described in the specific embodiments above can be combined in any suitable manner if not contradictory, and various not individually describe each possible combination.

Claims (9)

A step of dissolving a lipid in an organic solvent and removing the organic solvent to form a lipid film, wherein the lipid comprises lecithin and cholesterol, and the mass ratio of the lecithin and the cholesterol is 1:(0.05 ~ 0.35) ;
The lipid film obtained in step (1) is mixed with schizophyllan, a surfactant and a buffer solution and hydrated until the lipid film separates and forms a milky liquid to obtain a crude suspension of schizophyllan liposomes. a step, wherein the surfactant comprises a combination of polysorbate 80 and polyglyceryl-10 myristate, and the weight ratio of the polysorbate 80 and the polyglyceryl-10 myristate is (1.5-5):1; step (2);
and (3) sonicating the crude suspension of schizophyllan liposomes obtained in step (2) and filtering through a filtration membrane to obtain said schizophyllan liposomes. . the organic solvent according to step (1) comprises any one or a combination of at least two of methanol, ethanol, ethyl ether, acetone or dichloromethane;
The method for preparing schizophyllan liposomes according to claim 1 , characterized in that removing the organic solvent in step (1) uses a rotary evaporation method. The method for preparing schizophyllan liposomes according to claim 2, wherein the organic solvent contains ethyl ether and methanol, and the volume ratio of the ethyl ether and the methanol is (1-5):1. The method for preparing schizophyllan liposomes according to claim 1, wherein the buffer contains a phosphate buffer and has a pH of 6.5 to 7.5. The schizophyllan described in step (2) refers to a schizophyllan undiluted solution, and the schizophyllan undiluted solution is a β-glucan solution having a mass concentration of 0.5% to 5% obtained by fermentation of Suehirotake. A method for preparing schizophyllan liposomes according to claim 1 . The mass ratio of the schizophyllan stock solution and the lecithin is (0.01 to 0.25): 1,
The mass ratio of the schizophyllan stock solution and the surfactant is (2 to 10): 1,
The mass ratio of the schizophyllan stock solution and the buffer solution is 1: (5 to 20),
The method for preparing schizophyllan liposomes according to claim 5, characterized in that: The ultrasonic power of the ultrasonic treatment according to step (3) is 100 to 600 W,
The sonication time of the sonication according to step (3) is 20-40 min,
The filtration membrane of step (3) is a 0.45 μm micropore filtration membrane,
The particle size of the obtained schizophyllan liposomes according to step (3) is 50-200 nm,
The method for preparing schizophyllan liposomes according to any one of claims 1 to 5, characterized in that : a step (1) of dissolving the lecithin and the cholesterol in a mixed organic solvent of ethyl ether and methanol at a mass ratio of 1:(0.05 to 0.35), removing the organic solvent, and forming a lipid membrane;
The lipid film obtained in step (1) is mixed with a schizophyllan stock solution, a surfactant, and a phosphate buffer, and hydrated until the lipid film separates to form an opalescent liquid, yielding crude schizophyllan liposomes. obtaining a suspension, wherein the mass ratio of said schizophyllan stock solution and said lecithin is (0.01-0.25):1, and the mass ratio of said schizophyllan stock solution and said surfactant is (2-10): 1. Step (2), wherein the mass ratio of the schizophyllan stock solution and the phosphate buffer is 1: (5-20);
The crude suspension of said schizophyllan liposomes obtained in step (2) is sonicated at 100-600 W for 20-40 minutes and filtered through a 0.45 μm micropore filtration membrane to obtain said schizophyllan with a particle size of 50-200 nm. A method for preparing schizophyllan liposomes according to any one of claims 1 to 5 , characterized in that it comprises a step (3) of obtaining liposomes. A method for preparing a cosmetic containing schizophyllan liposomes, which comprises preparing schizophyllan liposomes by the method according to any one of claims 1 to 5.

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