CN116270262B

CN116270262B - Liposome solution, external preparation containing liposome solution for skin, and preparation method and application of external preparation

Info

Publication number: CN116270262B
Application number: CN202310310948.6A
Authority: CN
Inventors: 卢永杰; 张冬盈; 孙静; 张兵; 张炽坚; 艾勇; 何廷刚
Original assignee: Hua An Tang Biotech Group Co ltd
Current assignee: Hua An Tang Biotech Group Co ltd
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2024-01-02
Anticipated expiration: 2043-03-27
Also published as: CN116270262A

Abstract

The application discloses a liposome solution, an external preparation containing the liposome solution for skin, and a preparation method and application thereof. The preparation method of the liposome solution comprises the following steps: (1) Inoculating yeast into the red scenic spot water extract, fermenting, culturing and ultrasonic treating to obtain rhodiola rosea fermentation lysate; (2) Mixing 1 to 4 parts of rhodiola rosea fermentation lysate prepared in the step (1) with 15 to 25 parts of water, homogenizing, and homogenizing under high pressure to obtain the rhodiola rosea fermentation lysate, wherein the mixture comprises 1 to 0.05 to 0.125 part of lecithin, 0.3 to 0.8 part of cholesterol, 1 to 4.5 parts of polyglycerol emulsifier, 1 to 4.5 parts of polyalcohol and 0.075 to 0.5 part of glabridin. The composition containing the rhodiola rosea fermentation lysate and the glabridin has the effects of ideal antioxidation, whitening, brightening skin and the like, is simple in preparation process, improves the dispersibility of the glabridin an aqueous phase system, reduces cytotoxicity and irritation of the glabridin, and improves the use safety of products.

Description

Liposome solution, external preparation containing liposome solution for skin, and preparation method and application of external preparation

Technical Field

The application belongs to the technical field of fermentation, and particularly relates to a liposome solution, an external skin preparation containing the liposome solution, and a preparation method and application of the external skin preparation.

Background

Glabridin is a well-known whitening active ingredient, but has poor water solubility, and usually requires the addition of an emulsifier to improve the dispersibility in an aqueous phase, or the addition of a solubilizer to solubilize glabridin an aqueous phase, and has a complicated use method and limited improvement in dispersibility and solubility. Also, it has been shown that glabridin has a certain toxicity to melanocytes and human skin fibroblasts, and is highly irritating to skin and eyes, and when the glabridin content in cosmetics is increased, red blood streaks are easily caused to eyes. The water-insoluble, cytotoxic and irritating nature of glabridin limits the amount of glabridin added to cosmetics, which results in insignificant whitening effect.

Rhodiola rosea is a perennial herb or sub-shrub wild plant of rhodiola genus of rhodiola family rosaceae, and is named because the steep of flower, root, stem is red. Rhodiola rosea is also a traditional medicinal plant in China and is often used for preparing wine and medicinal preparations. The rhodiola rosea is reported to contain active ingredients such as rhodioside, aglycone tyrosol, cassavine and the like, and also contains active ingredients such as brass, coumarin, volatile oil, terpenes and polysaccharides, and has the effects of resisting fatigue, aging, virus, tumor, radiation and the like and improving the defensive power of a cardiovascular system and an immune system. However, the application research of rhodiola rosea in the cosmetic field is relatively few, and the extraction method of active ingredients which is suitable for rhodiola rosea and can be extracted from rhodiola rosea and has the beauty effect is high in use safety and has no stimulation to skin is an important difficult problem in the field facing to the numerous extraction processes.

Therefore, there is a need in the art to develop cosmetics containing glabridin with high safety in use, simple preparation process, stable system and remarkable whitening effect, which can efficiently utilize the beauty efficacy of rhodiola rosea and glabridin, and widen the application field of rhodiola rosea.

Disclosure of Invention

The technical problems to be solved by the application are to overcome the defects that the prior art has poor dispersion capability of the glabridin in water, low solubility, high cytotoxicity and irritation of the glabridin, limited improvement of the dispersion capability and solubilization effect of the glabridin in water phase by using the prior emulsifying agent or solubilizing agent, and the like, and the defects that the rhodiola rosea has less application in the field of cosmetics, and to provide a liposome solution, a skin external preparation containing the liposome solution, and a preparation method and application thereof. The composition containing the rhodiola rosea fermentation lysate and the glabridin has the effects of ideal antioxidation, whitening, brightening skin and the like, is simple in preparation process, improves the dispersibility of the glabridin an aqueous phase system, reduces cytotoxicity and irritation of the glabridin, and improves the use safety of products.

The application adopts the following technical scheme to solve the technical problems:

The application provides a preparation method of liposome solution, which specifically comprises the following steps:

(1) Inoculating yeast into the red scenic spot water extract, fermenting, culturing and ultrasonic treating to obtain rhodiola rosea fermentation lysate;

(2) Mixing 1 to 4 parts of lecithin, 0.05 to 0.125 part of cholesterol, 0.3 to 0.8 part of polyglycerol emulsifier, 1 to 4.5 parts of polyalcohol and 0.075 to 0.5 part of glabridin with 1 to 4 parts of rhodiola rosea fermentation lysate prepared in the step (1) and 15 to 25 parts of water, homogenizing, and homogenizing under high pressure to prepare the liposome solution.

In the step (1), the preparation method of the red scenic spot water extract can comprise the following steps: leaching dried rhodiola rosea and water for 1-3 h at 85-95 ℃, centrifuging and collecting supernatant.

In the preparation process of the red scenic spot water extract, the mass ratio of the dried rhodiola rosea to the water can be (1-5): 100.

in the preparation process of the rhodiola rosea water extract, the mesh number of the rhodiola rosea can be conventional in the field, preferably 10-100 meshes, and more preferably 40-50 meshes.

In the preparation process of the red scenic spot water extract, the leaching temperature is preferably 90-95 ℃.

In the preparation process of the red scenic spot water extract, the leaching time is preferably 1-3 h.

In the preparation process of the red scenic spot water extract, the rotation speed of the centrifugation can be 4000-5000 rpm, preferably 4000-4800 rpm.

In the preparation process of the red scenic spot water extract, the centrifugation time can be 25-40 min, preferably 30-35 min.

In the preparation process of the red scenic spot water extract, the operation of centrifugation can be followed by the operation of filtering the supernatant according to the conventional method in the field. The filter membrane used for the filtration may be conventional in the art, and may be typically 0.22 μm in pore size.

In the step (1), the rhodiola rosea water extract can also comprise sterilizing operation according to the conventional method in the field before use.

Wherein, the conditions and methods of the sterilization of the red scenic spot water extract can be conditions and methods conventional in the art for such operations, and can generally be high temperature sterilization.

When the sterilization is performed on the red scenic spot aqueous extract by the high temperature sterilization method, the sterilization temperature may be a temperature conventional in this kind of operation in the art, preferably 90 to 121 ℃, more preferably 110 to 121 ℃.

When the high temperature sterilization method is used to sterilize the red scenic spot water extract, the sterilization time may be a time conventional to such operations in the art, preferably 15 to 45 minutes, more preferably 30 to 40 minutes.

Wherein, after the sterilization operation is performed on the red scenic spot water extract, the red scenic spot water extract can be further cooled to room temperature according to the conventional technology in the art.

In the step (1), the yeasts may include at least one of "Saccharomyces cerevisiae purchased from China industry microbiological culture Collection center, with a accession number of CICC 1308", "Saccharomyces cerevisiae purchased from China industry microbiological culture collection center, with a accession number of CICC 1305", "Saccharomyces cerevisiae purchased from China industry microbiological culture collection center, with a accession number of CICC 1002", and "Saccharomyces cerevisiae purchased from China industry microbiological culture collection center, with a accession number of CICC 1346".

In step (1), the yeast may be used as yeast in accordance with conventional methods in the artThe yeast is added in the form of a bacterial liquid, and the concentration of the yeast in the yeast bacterial liquid can be 10 ⁶ ～10 ⁹ CFU/mL, preferably 10 ⁶ ～10 ⁷ CFU/mL。

In the step (1), the number of the yeasts inoculated in the red scenic spot water extract per unit volume can be 10 ⁵ ～10 ⁶ CFU/mL。

In step (1), the fermentation culture may be carried out in a shaker incubator as conventional in the art. Wherein the rotating speed of the shaking incubator can be 150-180 rpm.

In the step (1), the fermentation culture time may be 24 to 72 hours, preferably 48 to 72 hours.

In step (1), the temperature of the fermentation culture may be 25 to 35 ℃, preferably 25 to 28 ℃.

In step (1), the conditions and methods of the ultrasound may be conventional in the art, and the yeasts may generally be disrupted.

In step (1), the ultrasound may be performed in an ultrasound cytodisruption apparatus as is conventional in the art.

In step (1), the power of the ultrasound may be conventional in the art, and may generally be 65-130W.

In step (1), the time of the ultrasound may be a time conventional in the art for such operations, and may be generally 5 to 20 minutes, preferably 15 to 20 minutes.

In the step (1) of a preferred embodiment, the ultrasonic treatment adopts an intermittent ultrasonic treatment method, the ultrasonic treatment time is 5-20 s each time, the intermittent time is 10-20 s, and the sum of the ultrasonic treatment time and the intermittent time is 5-20 min. The intermittent time is the time interval between two adjacent ultrasound without ultrasound, as is conventional in the art.

In a more preferred embodiment, in step (1), the ultrasound is performed by intermittent ultrasound, each time the ultrasound is performed for 5 seconds, the intermittent time is 10 seconds, and the sum of the ultrasound time and the intermittent time is 10 minutes.

In the step (1), the operation of ultrasonic treatment may further include a sterilization operation.

Wherein the conditions and methods of sterilization may be conventional in the art and may generally be high temperature sterilization.

When the sterilization is performed on the material obtained after the ultrasonic treatment by the high-temperature sterilization method, the sterilization temperature may be a temperature which is conventional in the art for such an operation, preferably 90 to 121 ℃, more preferably 95 to 100 ℃.

When the high-temperature sterilization method is used to sterilize the material obtained after the ultrasonic treatment, the sterilization time may be a time conventional in the art, preferably 15 to 45 minutes, more preferably 30 to 40 minutes.

Wherein the sterilization operation may be followed by further cooling to room temperature as is conventional in the art.

In the step (2), the lecithin may include at least one of soybean lecithin, egg yolk lecithin and phosphatidylcholine type lecithin.

Wherein the phosphatidylcholine type lecithin may include dimyristoyl lecithin and/or dipalmitoyl phosphatidylcholine.

In the step (2), the polyglycerol-based emulsifier may include at least one of polyglycerol-10 stearate, polyglycerol-10 laurate, polyglycerol-10 myristate, polyglycerol-10 palmitate and polyglycerol-10 oleate.

In step (2), the polyol may comprise at least one of dipropylene glycol, glycerin, propylene glycol, butylene glycol, and pentylene glycol, preferably dipropylene glycol.

In the step (2), the weight part of the cholesterol is preferably 0.1 to 0.125 part.

In the step (2), the weight part of the polyglycerol-based emulsifier is preferably 0.5 to 0.8 part.

In the step (2), the weight part of the polyol is preferably 2.5 to 3.5 parts.

In the step (2), the glabridin is preferably 0.075 to 0.25 parts by weight, more preferably 0.125 to 0.25 parts by weight.

In the step (2), the rhodiola rosea fermentation lysate is preferably 2-4 parts by weight.

In the step (2), the water is preferably 19 to 25 parts by weight.

In the step (2), a preservative can be further added in the mixing process.

Wherein, the weight portion of the preservative can be 0.25 to 2 portions, preferably 0.5 to 1 portion.

Among them, the preservative may include an alcohol preservative and/or p-hydroxyacetophenone conventionally used in the cosmetic field. The alcohol preservative may comprise at least one of octanediol, 1, 2-hexanediol, and phenoxyethanol, and preferably comprises octanediol and 1, 2-hexanediol. When the preservative comprises octanediol and 1, 2-hexanediol, the mass ratio of octanediol to 1, 2-hexanediol may be 1: (0.5 to 2), preferably 1:1.

In step (2), the preparation method of the mixture may include the steps of: the lecithin, the cholesterol, the polyglycerol emulsifier, the polyol and the glabridin are mixed.

Wherein the temperature of the mixing may be 70 to 80 ℃, preferably 75 to 80 ℃.

Wherein the mixing time may be conventional in the art, and typically the components are dissolved in their entirety.

In step (2), the temperature of the homogenization may be from 70 to 80 ℃, preferably from 75 to 80 ℃.

In the step (2), the homogenizing time may be 5 to 15 minutes.

In step (2), the homogenization may be carried out in a homogenizer as conventional in the art, and the rotational speed of the homogenizer may be 5000 to 10000rpm, preferably 7000 to 8000rpm.

In step (2), the high-pressure homogenization may be carried out at a pressure of 200 to 800bar, preferably 600 to 800bar.

In the step (2), the time of high-pressure homogenization can be 5-10 min.

In step (2), the high pressure homogenization may be performed in a high pressure homogenizer as conventional in the art.

The present application also provides a liposome solution prepared by the method for preparing a liposome solution as described above.

The application also provides an application of the liposome solution in preparing external skin preparations, wherein the liposome solution is directly used as a product, an additive or a substrate.

In some embodiments, the liposome solution may be used as at least one of an antioxidant active ingredient, a whitening active ingredient, and a skin gloss improving active ingredient in the skin external agent.

The present application also provides a skin external agent comprising the liposome solution as described above.

In some embodiments, the external preparation for skin may further include an active ingredient conventionally used in the art, and may generally include at least one of a humectant, an emollient, a preservative, a thickener, a pH adjuster, a chelating agent, and a solvent.

Wherein the humectant may comprise a polyol humectant and/or betaine. The polyhydric alcohol humectant may include at least one of butylene glycol, glycerin, and 1, 2-hexanediol.

Wherein the emollient may comprise at least one of polydimethylsiloxane, caprylic/capric triglyceride, and glyceryl caprylate.

Wherein the preservative may comprise phenoxyethanol.

Wherein the thickener may comprise acrylic acid (esters) based/C10-30 alkanol acrylate cross-linked polymers.

Wherein the pH regulator may comprise arginine

Wherein the chelating agent may comprise disodium EDTA.

Wherein the solvent may comprise deionized water.

Wherein the mass ratio of the humectant to the liposome solution may be conventional in the art, preferably (4 to 7): 1, more preferably (5 to 6): 1.

wherein the mass ratio of said emollient to said liposome solution may be conventional in the art, preferably (0.5 to 1.5): 1, more preferably (0.7 to 1): 1, for example 0.85:1.

wherein the mass ratio of the preservative to the liposome solution may be conventional in the art, preferably (0.1-0.2): 1, for example 0.15:1.

wherein the mass ratio of the thickener to the liposome solution may be conventional in the art, preferably (0.05 to 0.15): 1, for example 0.1:1.

wherein the mass ratio of the pH adjuster to the liposome solution may be conventional in the art, preferably (0.05 to 0.15): 1, for example 0.1:1.

wherein the mass ratio of the chelating agent to the liposome solution may be conventional in the art, preferably (0.02-0.05): 1, for example, 0.025:1.

wherein the mass ratio of the solvent to the liposome solution may be conventional in the art, preferably (35-45): 1, preferably 42.125:1.

in some embodiments, the skin external preparation may include, but is not limited to, a mask, a serum, or a toner, as is conventional in the art.

In some embodiments, the liposome solution may comprise 1% to 20%, preferably 1% to 5% of the skin external agent by mass.

In a preferred embodiment, the skin external preparation comprises 1 part of the liposome solution as described above, 5.65 parts of humectant, 0.85 parts of emollient, 0.15 parts of preservative, 0.1 parts of thickener, 0.2 parts of pH regulator, 0.025 parts of chelating agent and 42.125 parts of solvent.

In a more preferred embodiment, the skin external preparation comprises 1 part of the liposome solution as described above, 2.5 parts of butanediol, 1.5 parts of glycerol, 1.5 parts of betaine, 0.15 part of 1, 2-hexanediol, 0.5 part of polydimethylsiloxane, 0.25 part of caprylic/capric triglyceride, 0.1 part of caprylic glyceride, 0.15 part of phenoxyethanol, 0.1 part of acrylic acid (esters) type/C10-30 alkanol acrylate cross-linked polymer, 0.1 part of arginine and 0.025 part of disodium EDTA.

In some embodiments, the room temperature generally refers to 18-30 ℃.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the application.

Reagents and starting materials for the present application are commercially available.

The positive progress effect of this application lies in: the composition containing the rhodiola rosea fermentation lysate and the glabridin has the effects of resisting oxidation, whitening skin, improving skin glossiness and the like, the cosmetic effects of the rhodiola rosea and the glabridin are fully utilized, the preparation process is simple, the dispersing capability of the glabridin an aqueous phase system is improved, the cytotoxicity and the irritation of the glabridin are reduced, and the use safety of the product is improved.

Drawings

The present disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are included to provide a further illustration of the preferred embodiments of the disclosure and to explain the principles and advantages of the disclosure, are incorporated in and form a part of the specification along with the detailed description that follows.

Wherein:

FIG. 1 is a graph showing the comparison of erythrocyte hemolysis rates of the products obtained in examples 1 to 6 and comparative examples 1 to 7;

FIG. 2 is a graph showing the DPPH radical scavenging ability of the products obtained in examples 1 to 6 and comparative examples 1 to 7;

FIG. 3 is a graph showing comparison of tyrosinase activity inhibition rates of the products prepared in examples 1 to 6 and comparative examples 1 to 7;

FIG. 4 is a graph showing the comparison of the viability of HaCat cells after the treatment of HaCat cells with the products prepared in examples 1 to 6 and comparative examples 1 to 7;

FIG. 5 is a comparative graph showing the melanin production-inhibiting ability of the products obtained in examples 1 to 6 and comparative examples 1 to 7;

fig. 6 is a graph showing the improvement of skin glossiness with time of the essence prepared in application example 1.

Detailed Description

The present application is further illustrated by way of examples below, but is not thereby limited to the scope of the examples described. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The preparation method of the saccharomycete liquid in the following examples and comparative examples comprises the following steps:

(1) Activation of saccharomyces cerevisiae strains: preparing YPD liquid culture medium (1% (w/v) and accurately weighing peptone, glucose and yeast extract powder according to the proportion of 2% to 1%), autoclaving at 121deg.C for 30min, cooling to room temperature after sterilization, taking out the Saccharomyces cerevisiae strain to be activated from the refrigerator at-80deg.C after ultraviolet sterilization, melting at room temperature, inoculating into YPD liquid culture medium, and placing into a shaking table at 28deg.C and 180rpm for strain activation.

(2) Purifying saccharomyces cerevisiae strains: activated Saccharomyces cerevisiae was diluted in a gradient and plated to obtain single colonies.

(3) Expansion culture of Saccharomyces cerevisiae strain: the purified single Saccharomyces cerevisiae strain is inoculated into YPD liquid culture medium, cultured in a shaking table at 28 ℃ and 180rpm, the absorbance at 600nm is measured, and when the absorbance is 1.2, the strain is in logarithmic growth phase, and the strain can be inoculated and cultured at the moment.

Example 1

(1) Crushing dried rhodiola rosea, sieving with a 50-mesh sieve, mixing with water, preparing according to the proportion of the rhodiola rosea accounting for 5% of the water by mass percent, extracting for 1h at 90 ℃, centrifuging for 30min at 4800rpm after extraction, filtering the supernatant obtained after centrifugation with a 0.22 mu m filter membrane to obtain rhodiola rosea water extract, sterilizing the rhodiola rosea water extract at 121 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate;

(2) Saccharomyces cerevisiae is purchased from China industry microbiological culture collection center (CICC), with accession number CICC1308; preparing saccharomyces cerevisiae bacteria liquid according to the method, inoculating the saccharomyces cerevisiae bacteria liquid into the fermentation substrate prepared in the step (1), wherein the concentration of the inoculated saccharomyces cerevisiae bacteria in the unit volume of the fermentation substrate is 10 ⁶ Fermenting and culturing CFU/mL at 28deg.C for 72 hr with shaking incubator at rotation speed of 180rpm, and transferring the obtained material into ultrasonic cell disruption instrument for ultrasoundCrushing saccharomycetes, adopting an intermittent ultrasonic mode, carrying out ultrasonic treatment for 5 seconds at intervals of 10 seconds, wherein the total time of the intervals and the ultrasonic treatment is 10 minutes, the ultrasonic power is 130W, and sterilizing for 30 minutes at 95 ℃ after ultrasonic treatment; sterilizing, and cooling to room temperature to obtain radix Rhodiolae fermented lysate;

(3) Mixing 4g of soybean lecithin, 0.5g of cholesterol, 2g of polyglycerol-10 stearate, 10g of dipropylene glycol, 1g of octylglycol, 1g of 1, 2-hexanediol and 1g of glabridin, and heating to 75 ℃ to dissolve all the components; adding 8g of rhodiola rosea fermentation lysate prepared in the step (2) and 72.5g of water into the system, carrying out heat preservation and homogenization for 15min at the temperature of 75 ℃, rotating the homogenizer at 7000rpm, transferring the mixed solution into a high-pressure homogenizer, carrying out high-pressure homogenization for 10min, and obtaining the liposome solution at the pressure of 800 bar.

Example 2

The difference compared with example 1 is that the fermentation broth in step (2) is different, specifically, the Saccharomyces cerevisiae with accession number CICC1308 is replaced by equal amount of Saccharomyces cerevisiae purchased from China industry microbiological culture Collection center (CICC), and the accession number CICC 1305 is the same as in example 1.

Example 3

The difference compared with example 1 is that the fermentation broth in step (2) is different, specifically, the Saccharomyces cerevisiae with accession number CICC1308 is replaced with equal amount of Saccharomyces cerevisiae purchased from China industry microbiological culture Collection center (CICC), and the preparation method has the same preparation method as that of example 1 except that the fermentation broth with accession number CICC 1002 is replaced with equal amount of Saccharomyces cerevisiae.

Example 4

The difference compared with example 1 is that the fermentation broth in step (2) is different, specifically, the Saccharomyces cerevisiae with accession number CICC1308 is replaced by equal amount of Saccharomyces cerevisiae purchased from China industry microbiological culture Collection center (CICC), and the Saccharomyces cerevisiae with accession number CICC 1346, and other conditions and parameters are the same as those in example 1.

Example 5

The difference compared with example 1 is only that the addition amounts of glabridin and water in step (3) are different; in this example, the amount of glabridin added was 0.5g, the amount of water added was 73g, and the other conditions were the same as in example 1.

Example 6

The difference compared with example 1 is only that the addition amounts of glabridin and water in step (3) are different; in this example, the amount of glabridin added was 0.3g, the amount of water added was 73.2g, and the other conditions were the same as in example 1.

Application example 1

The essence is prepared by mixing the components in table 1 uniformly.

TABLE 1

Comparative example 1

Compared with example 1, the method mainly comprises the steps of replacing Saccharomyces cerevisiae with the preservation number of CICC1308 with an equal amount of lactobacillus, selecting bifidobacterium longum with the preservation number of CICC 6207 from China center for type culture collection (CICC), and correspondingly adjusting fermentation culture conditions, wherein the method specifically comprises the following steps:

(2) The lactobacillus is selected from Bifidobacterium longum with a deposit number of CICC 6207 purchased from China industry microbiological culture Collection center (CICC); inoculating purified Bifidobacterium longum into MRS culture medium, standing at 37deg.C for culturing to obtain lactobacillus solution, inoculating lactobacillus solution into fermentation substrate prepared in step (1), wherein the amount of lactobacillus inoculated per unit volume of fermentation substrate is 10 ⁶ CFU/mL, standing at 35deg.C for 72 hr, fermenting, and culturing to obtain the final productTransferring to an ultrasonic cell disruption instrument for ultrasound for disrupting yeast, and performing ultrasound at intervals of 10s for 5s with total time of 10min, power of ultrasound of 130W, and sterilizing at 95deg.C for 30min; sterilizing, and cooling to room temperature to obtain radix Rhodiolae fermented lysate;

Comparative example 2

The difference compared to example 1 is only that the operation of step (3) is not performed, specifically: 8g of the rhodiola rosea fermentation lysate prepared in the step (2) in the example 1 is mixed with 92g of water.

Comparative example 3

Compared with example 1, the only difference is that the operations of step (1) and step (2) are not performed, and the rhodiola rosea fermentation lysate prepared in step (2) is replaced by the same amount of water in step (3), and the method specifically comprises the following steps:

mixing 4g of soybean lecithin, 0.5g of cholesterol, 2g of polyglycerol-10 stearate, 10g of dipropylene glycol, 1g of octylglycol, 1g of 1, 2-hexanediol and 1g of glabridin, and heating to 75 ℃ to dissolve all the components; then 80.5g of water is added into the system, the temperature is kept for homogenization for 15min at 75 ℃, the rotation speed of a homogenizer is 7000rpm, the mixed solution is transferred into a high-pressure homogenizer, the high-pressure homogenization is carried out for 10min, and the high-pressure homogenization pressure is 800bar, so that liposome solution is obtained.

Comparative example 4

Preparing an aqueous solution of glabridin: 1g of glabridin is weighed and dispersed in 10g of 1, 3-propanediol, ultrasonic treatment is carried out for 1h at 25 ℃, and 89g of deionized water is added to prepare 1% glabridin water solution by mass percent.

Comparative example 5

Compared with example 1, the difference is that in the step (3), no glabridin is added, namely 1g of glabridin is replaced by 1g of water, and other condition parameters are the same as those in example 1, and the method specifically comprises the following steps:

(2) Saccharomyces cerevisiae is purchased from China industry microbiological culture collection center (CICC), with accession number CICC 1308; preparing saccharomyces cerevisiae bacteria liquid according to the method, inoculating the saccharomyces cerevisiae bacteria liquid into the fermentation substrate prepared in the step (1), wherein the concentration of the inoculated saccharomyces cerevisiae bacteria in the unit volume of the fermentation substrate is 10 ⁶ Fermenting and culturing CFU/mL for 72h at 28 ℃, rotating the shaking incubator at 180rpm, transferring the prepared material into an ultrasonic cell disruption instrument for ultrasound after fermentation and culture are finished, disrupting microzyme, adopting an intermittent ultrasound mode, performing ultrasound for 5s at intervals of 10s, wherein the total time of the interval and the ultrasound is 10min, the power of the ultrasound is 130W, and sterilizing for 30min at 95 ℃ after ultrasound; sterilizing, and cooling to room temperature to obtain radix Rhodiolae fermented lysate;

(3) Mixing 4g of soybean lecithin, 0.5g of cholesterol, 2g of polyglycerol-10 stearate, 10g of dipropylene glycol, 1g of octylglycol and 1g of 1, 2-hexanediol, and heating to 75 ℃ to dissolve all the components; adding 8g of rhodiola rosea fermentation lysate prepared in the step (2) and 73.5g of water into the system, carrying out heat preservation and homogenization for 15min at the temperature of 75 ℃, rotating the homogenizer at 7000rpm, transferring the mixed solution into a high-pressure homogenizer, carrying out high-pressure homogenization for 10min, and obtaining the liposome solution at the pressure of 800 bar.

Comparative example 6

The difference from example 1 is that the amount of glabridin and water added in step (3) was 3g, the amount of glabridin added was 70.5g, and the other conditions were the same as in example 1.

Comparative example 7

Compared with the embodiment 1, the difference is that the ultrasonic treatment is not performed in the step (2) to prepare the rhodiola fermentation product filtrate, the rhodiola fermentation lysate added in the step (3) is replaced by the rhodiola fermentation product filtrate with the same amount, and other condition parameters are the same as those in the embodiment 1, and the specific operation method is as follows:

(2) Saccharomyces cerevisiae is purchased from China industry microbiological culture collection center (CICC), with accession number CICC 1308; preparing saccharomyces cerevisiae bacteria liquid according to the method, inoculating the saccharomyces cerevisiae bacteria liquid into the fermentation substrate prepared in the step (1), wherein the concentration of the inoculated saccharomyces cerevisiae bacteria in the unit volume of the fermentation substrate is 10 ⁶ Fermenting and culturing CFU/mL at 28deg.C for 72h, rotating at 180rpm, and sterilizing at 95deg.C for 30min after fermentation and culture; sterilizing, cooling to room temperature, centrifuging at 4800rpm for 30min, collecting supernatant, filtering with 0.22 μm filter membrane, and collecting filtrate to obtain radix Rhodiolae fermentation product filtrate;

(3) Mixing 4g of soybean lecithin, 0.5g of cholesterol, 2g of polyglycerol-10 stearate, 10g of dipropylene glycol, 1g of octylglycol, 1g of 1, 2-hexanediol and 1g of glabridin, and heating to 75 ℃ to dissolve all the components; adding 8g of the rhodiola fermentation product filtrate obtained in the step (2) and 72.5g of water into the system, carrying out heat preservation and homogenization for 15min at the temperature of 75 ℃, rotating the homogenizer at the speed of 7000rpm, transferring the mixed solution into a high-pressure homogenizer, carrying out high-pressure homogenization for 10min, and obtaining the liposome solution at the pressure of 800 bar.

Effect example 1 System stability data

The particle sizes of the products obtained in examples 1 to 6, comparative example 1, comparative example 3 and comparative examples 5 to 7 were measured, and the results are shown in Table 2. The products prepared in comparative examples 2 and 4 were solutions, and the particle size could not be measured.

TABLE 2 particle size test experiment results

Numbering device	Particle size (nm)
		Example 1	102
Example 2	109
		Example 3	104
Example 4	105
		Example 5	99.1
Example 6	88.5
		Comparative example 1	116
Comparative example 3	109
		Comparative example 5	108
Comparative example 6	2885
		Comparative example 7	105

Research shows that the film permeability of the nano particles is reduced along with the increase of the particle size, and when the particle size of the product is within 100nm, the nano particles have high film permeability; the particles having a particle diameter in the range of 100 to 200nm also have a higher film permeability, but the film permeability is slightly lower than that of a product having a particle diameter of 100nm or less. The particle size threshold of the passive transport membrane is 500nm, and particles larger than 500nm are difficult to enter the circulatory system across polar epithelial cells.

The liposome prepared in the embodiments 1 to 6 has smaller particle size, less than 200nm, good film permeability, easy absorption by skin and high system stability. The particle size of the product prepared in comparative example 5 is 2885nm, and it is clear that when the glabridin content is higher than the limit range of the application, the particle size of the assembled material in the system is larger, the film permeability is poor, and the glabridin is not easy to be absorbed by skin.

Effect example 2 irritation experiment

1. Preparation of Phosphate Buffer (PBS): ph=7.2, and was used up within one week after formulation.

2. Sample configuration to be tested: the products prepared in the examples or comparative examples were diluted with deionized water to a volume percent of 25% of the liquid to be tested.

3. Preparation of RBC suspension

Preparation of erythrocyte suspension (RBC)

Transferring the blood cell sediment in the centrifuge tube to a new EP tube by using a disposable straw, taking 1mL of RBC sediment in 15mL of EP tube, adding 9mL of PBS buffer for 10-time dilution, then gently shaking and mixing uniformly, centrifuging at 1500rpm for 5min, pouring out the supernatant, repeatedly cleaning for three times until PBS in the tube is colorless, and adding 10mL of PBS buffer to obtain the red blood cell suspension.

4. Experimental group setup

Total hemolysis control group: 0.75mL of water+0.25 mL of red blood cell suspension;

negative control group: 0.75mL of PBS buffer+0.25 mL of red blood cell suspension;

sample group: 0.75mL of the test solution+0.25 mL of the red blood cell suspension;

sample control: 0.75mL of test solution+0.25 mL of PBS;

according to the proportion, the total volume is 1mL, the EP tube is placed in 150rpm,37 ℃ in a shaking table for 1h, then each EP tube is placed in a centrifuge, the centrifugation is carried out at 10000 Xg speed for 1min to stop the cultivation, the supernatant is taken after the centrifugation, the absorbance is measured at 560nm, and the data is recorded and saved.

The hemolysis rate of the sample group was calculated according to the following formula, and the results are shown in table 3 and fig. 1 (in table 3 and fig. 1, "×" indicates a significant difference from example 1, P <0.05; "×" indicates an extremely significant difference from example 1, P < 0.01).

Erythrocyte hemolysis rate of sample group =

(A _{Sample group} -A _{Sample control} -A _{Negative control group} )/(A _{Total hemolysis control group} -A _{Negative control group} )×100％。

TABLE 3 erythrocyte hemolysis ratio test results

Numbering device	Erythrocyte hemolysis rate
		Example 1	3.30％
Example 2	3.41％
		Example 3	3.21％
Example 4	3.15％
		Example 5	2.98％
Example 6	2.84％
		Comparative example 1	4.75％*
Comparative example 2	10.80％**
		Comparative example 3	16.62％**
Comparative example 4	42.14％**
		Comparative example 5	3.24％
Comparative example 6	56.04％**
		Comparative example 7	8.44％**

The results show that: the red blood cells prepared in examples 1 to 6 of the present application have low hemolysis rate and low irritation.

The product prepared in example 1 has significantly lower irritation than that of comparative example 1, presumably because lactic acid bacteria used in comparative example 1 secrete acidic substances such as lactic acid during fermentation, so that erythrocyte membrane protein denaturation causes chemical hemolysis.

Comparative example 4 produced an aqueous glabridin solution having a significantly higher hemolysis rate than example 1 (p <0.01,); it can be seen that although the organic solvent can improve the dispersibility of glabridin in water after the dissolution, the dispersion system still has extremely remarkable irritation.

Comparative example 3 is different from example 1 in that the red blood cell hemolysis rate of glabridin is reduced to some extent by only coating glabridin with soybean lecithin or the like without adding rhodiola rosea fermentation lysate, but the red blood cell hemolysis rate is still extremely higher than that of example 1. Therefore, the combination of the rhodiola rosea fermentation lysate and the liposome containing the glabridin, the soybean lecithin and other substances can further remarkably improve the dispersion capacity of the glabridin in water and the stability of the system, reduce the content of the non-coated glabridin in the system and further reduce the irritation of the system.

Comparative example 6 has an excessively high amount of glabridin compared with examples 1 to 6, and the amount of glabridin coated in the final product is limited, and a large amount of free and uncoated glabridin is present in the system, which results in a higher erythrocyte hemolysis rate.

Comparative example 7 differs from example 1 only in that the rhodiola rosea fermentation lysate is replaced with an equivalent amount of licorice fermentation product filtrate, and the product obtained in comparative example 7 has a limited reduction degree of erythrocyte hemolysis compared with comparative example 3, whereas example 1 is more advantageous. It is presumed that the rhodiola rosea fermentation lysate used in example 1 contains more substances which are favorable for the dispersion of the glabridin and improve the stability of the system, thereby reducing the content of the non-coated glabridin in the system and reducing the irritation of the system.

Effect example 3 antioxidant experiment (DPPH radical scavenging Rate)

DPPH is an early synthetic organic radical, commonly used to evaluate the hydrogen donating ability of antioxidants, which is very stable in organic solvents, purple in color, and has a characteristic absorption peak at 517nm, when a radical scavenger is encountered, the lone pair of electrons of DPPH are paired to fade it, i.e., the absorbance at the maximum absorption wavelength becomes small. Therefore, the effect of the sample on DPPH radical scavenging can be evaluated by measuring the change in absorbance.

Preparing a liquid to be tested: the products prepared in the above examples or comparative examples were each formulated as 10% volume percent of the liquid to be tested using deionized water.

The DPPH free radical scavenging experiment comprises the following specific experimental steps:

(1) Mixing an equal volume (1 mL) of the solution to be tested with 0.8mg/mL of DPPH solution uniformly (A1 pipe);

(2) Mixing equal volume (1 mL) of absolute ethanol with 0.8mg/mL of DPPH solution uniformly (A2 tube);

(3) Mixing the same volume (1 mL) of absolute ethanol with the liquid to be measured (A) ₃ A tube);

(4) After reaction in the dark for 30min, A was measured at 517nm ₁ Tube A ₂ Tube A ₃ Tube absorbance values; the clearance rate calculation formula is: DPPH radical scavenging = [ (a) ₂ +A ₃ )-A ₁ ]/A ₂ ×100％。

The DPPH radical scavenging results are shown in table 4 and fig. 2 (in table 4 and fig. 2, "x" indicates that there is a very significant difference, P < 0.01), compared to example 1.

TABLE 4 DPPH radical scavenger test results

The results show that: the products prepared in examples 1-6 of this application have desirable DPPH radical scavenging, i.e., desirable antioxidant, capabilities.

The DPPH radical scavenging rate in example 1 was very significantly higher than in comparative example 1. Therefore, compared with the liposome solution prepared by taking the rhodiola rosea fermentation lysate as the raw material, the liposome solution prepared by taking the rhodiola rosea fermentation lysate as the raw material has more advantages in antioxidation effect.

As can be seen from the results of example 1 and comparative example 2, comparative example 3 and comparative example 5, the addition amount of rhodiola rosea fermentation lysate in the present application is relatively small, and the DPPH radical scavenging rate per se is relatively weak (see comparative example 2). And the product (see comparative example 5) prepared by mixing the rhodiola rosea fermentation lysate with liposome containing soybean lecithin and the like is not significantly improved compared with the rhodiola rosea fermentation lysate (see comparative example 2). However, when the rhodiola rosea fermentation lysate is combined with glabridin and liposome including soybean lecithin and the like, DPPH free radical clearance rate of a system can be remarkably improved, and the three components have synergistic promotion effect on oxidation resistance after being combined, namely, the DPPH free radical clearance rate (83.27%) of the product prepared in the example 1 is higher than the sum of the DPPH free radical clearance rates of the products prepared in the comparative example 2 (13.47%) and the comparative example 3 (55.36%) by 14.44%.

Example 1 produced a product with higher DPPH radical scavenging than comparative example 7. Presumably, the antioxidant effect of the lysate is more advantageous because it contains bacterial components in addition to the metabolites; in addition, the lysate contains substances which are more favorable for uniformly dispersing the glabridin in water and improve the amphiphilicity of the glabridin in the system, thereby showing more excellent antioxidant capacity.

Effect example 4 tyrosinase inhibition assay

PBS buffer with pH value of 6.8 was prepared.

Tyrosinase solution: tyrosinase was prepared as 100u/mL enzyme solution using PBS phosphate buffer at pH 6.8. (tyrosinase powder activity is not less than 1000 unit/mg)

0.1M HCl solution: 0.431mL of HCl solution with 36-38% of HCl content is used, and distilled water is used for constant volume to 50mL.

0.5mg/mL levodopa solution: 0.02g of L-dopa is taken and dissolved in 14mL of 0.1mol/L HCl solution, 26mL of PBS phosphate buffer solution with pH value of 6.8 is added, and the mixture is stirred and mixed uniformly for 40mL.

Sample configuration to be tested: the products prepared in the examples or comparative examples were diluted with deionized water to give 30% volume percent of the liquid to be tested.

Negative control group: water;

blank group: PBS buffer (ph=6.8);

Each set of reaction solutions was prepared according to Table 5.

Table 5 proportion of components in each group of reaction liquid and preparation method

Referring to Table 5, a 1.5mL EP tube was used to set up sample tube (T), sample background (T ₀ ) Enzyme reaction tube (C) and solvent background (C) ₀ ) The sample tube (T) of each tested concentration of each sample needs to be provided with 3 parallel tubes, and the enzyme reaction tube (C) needs to be provided with 3 parallel tubes.

In the sample tube (T) and sample background (T ₀ ) Each of which was added 250. Mu.L of the same concentration of the sample solution, the enzyme reaction tube (C) and the solvent background (C) ₀ ) Then 250. Mu.L of PBS phosphate buffer was added, respectively.

125. Mu.L of tyrosinase solution was added to each of the sample tube (T) and the enzyme reaction tube (C), and the sample background (T) ₀ ) With solvent background (C) ₀ ) The sample and tyrosinase were thoroughly mixed with 125. Mu.L PBS phosphate buffer instead, and incubated in a 37℃water bath for 10min. To each tube was added 500. Mu.L of a levodopa solution in sequence, the reaction time was controlled to 5 minutes for each tube, and each tube of the reaction solution was immediately transferred into a cuvette, absorbance was measured at 475nm, and recorded. Substituting into the formula to obtain tyrosinase activity inhibition rate of sample to be tested after dilution of the product obtained in the above example or comparative example, wherein the results are shown in Table 6 and FIG. 3 (in Table 6 and FIG. 3, "x" represents significant difference compared with example 1, P) <0.05；)。

Tyramine as sample to be testedInhibition of the Activity of the acid enzyme = [ (C-C) ₀ )-(T-T ₀ )]/(C-C ₀ )×100％。

TABLE 6 results of tyrosinase activity inhibition assay

Numbering device	Tyrosinase activity inhibition rate
		Example 1	97.87％
Example 2	90.89％
		Example 3	92.51％
Example 4	85.02％
		Example 5	92.31％
Example 6	86.94％
		Comparative example 1	78.14％*
Comparative example 2	44.13％*
		Comparative example 3	33.60％*
Comparative example 4	75.40％*
		Comparative example 5	59.72％*
Comparative example 6	63.77％*
		Comparative example 7	78.54％*

The results show that: the products prepared in examples 1-6 of the application have ideal tyrosinase activity inhibition rate, and can be seen to have ideal whitening effect.

Comparative example 1 the tyrosinase activity inhibition rate was relatively low compared to example 1. Therefore, the whitening effect of the liposome solution prepared by taking the yeast rhodiola rosea fermentation lysate as the raw material is better than that of the liposome solution prepared by taking the lactic acid bacteria rhodiola rosea fermentation lysate as the raw material.

As can be seen from the effect data of comparative example 2, comparative example 3 and example 1, the addition amount of rhodiola rosea fermentation lysate in the present application is relatively small, and it has a certain whitening effect by itself, but is relatively weak (see comparative example 2). However, when the rhodiola rosea fermentation lysate is combined with glabridin and liposome containing soybean lecithin and the like, the tyrosinase activity inhibition rate of the system can be obviously improved, and the three components are combined to have synergistic promotion effect on the whitening effect, namely, the tyrosinase activity inhibition rate (97.87%) of the product prepared in the example 1 is 20.14% higher than the sum of the tyrosinase inhibition rates of the products prepared in the comparative example 2 (44.13%) and the comparative example 3 (33.60%).

According to the experimental result of comparative example 4, it is known that the aqueous solution of glabridin after the co-dissolution of the organic solvent has an ideal tyrosinase inhibitory activity, which indicates that glabridin does have an excellent in vitro whitening effect. In comparative example 3, the rhodiola rosea fermentation lysate is not added, and only liposome including soybean lecithin and the like is used for wrapping the glabridin, so that although the solubility and the dispersion capacity of the glabridin in an aqueous phase can be improved, the whitening activity of the glabridin is weakened to a certain extent, and the glabridin is not easy to release after being wrapped by liposome including soybean lecithin and the like, so that the glabridin cannot play a role in a system.

Comparative example 6 was unstable due to the excessively high content of glabridin in the system, and the glabridin was poorly dispersed in the system, and self-aggregation occurred, which lowered the bioavailability thereof, and thus resulted in a remarkably lower tyrosinase inhibition rate than that of the product prepared in example 1.

The tyrosinase inhibition rate of the product prepared in example 1 was significantly increased compared to comparative example 7. According to the result, compared with the product prepared in the comparative example 7, the product prepared in the example 1 is presumed to be more beneficial to improving the solubility and dispersion stability of the glabridin in the system by combining the rhodiola rosea fermentation lysate with soybean lecithin and other substances, and the bioavailability of the glabridin is improved to a certain extent while the irritation of the glabridin is reduced, so that the whitening effect of the system is improved.

Effect example 5CCK-8

Taking out culture flask containing HaCat cells (human immortalized epidermal cells) with good growth state from the incubator, washing with PBS buffer solution with pH value of 7.4 twice, adding 0.5mL pancreatin into each flask, placing into the cell incubator for 2min, waiting for complete digestion and wall suspension of cells, adding 1mL DMEM medium with serum to stop pancreatin digestion, transferring cell suspension into 15mL centrifuge tube, centrifuging at 1500rpm for 5 min, discarding supernatant, and blowing cell precipitate with DMEM medium with serumEven, then transfer to T25 flask at 5% CO ₂ Subculturing in an incubator at 37 ℃ for two to three days, and carrying out the subsequent experiments until the cell fusion rate is more than 80%.

Will be 8×10 ³ Inoculating the individual cells/100 mu L of the culture medium into a 96-well plate for culturing for 24 hours, and sucking and removing the original culture medium; the test group was treated with 100. Mu.L of the sample to be tested (the sample prepared in the above example or comparative example was diluted with serum-free DMEM medium to prepare a sample to be tested having a volume percentage of 0.03%) for 24 hours, 10. Mu.L of CCK-8 reagent was added to each well, incubation was continued at 37℃for 1 hour, and the absorbance OD at an absorption wavelength of 450nm was measured _{Sample to be measured} . Adding 10 μl of serum-free DMEM medium into each well of control group for 24 hr, adding 10 μl of CCK-8 reagent into each well, incubating at 37deg.C for 1 hr, and measuring absorbance OD at absorption wavelength of 450nm _Control 。

Cell viability = OD _{Sample to be measured} /OD _{Control group} 100% and the cell viability of the test sample treated cells prepared in the above examples or comparative examples was calculated according to the formula, and the results are shown in Table 7 and FIG. 4 (in Table 7 and FIG. 4, ".+ -. Indicates a significant difference, P, from example 1)<0.05; "X" means that there is a very significant difference, P, from example 1<0.01)。

TABLE 7 cell viability assay results

Numbering device	Cell viability
		Example 1	93.62％
Example 2	82.45％
		Example 3	84.05％
Example 4	72.90％
		Example 5	97.10％
Example 6	96.36％
		Comparative example 1	80.23％**
Comparative example 2	93.28％
		Comparative example 3	70.28％**
Comparative example 4	60.48％**
		Comparative example 5	99.45％*
Comparative example 6	72.36％**
		Comparative example 7	80.52％**

The results show that: the products prepared in examples 1-6 of the application have significantly reduced HaCat cytotoxicity and low skin irritation.

The cytotoxicity of the product obtained in example 1 was significantly lower than that of comparative example 1 (p < 0.01), presumably because the lactic acid bacteria used in comparative example 1 secrete acidic substances such as lactic acid during fermentation, which affects cell viability.

Comparative example 4 the cytotoxicity of the aqueous glabridin solution was significantly higher than that of example 1 (p <0.01,); it can be seen that although the organic solvent can improve the dispersibility of glabridin in water after the dissolution, the dispersion system still has extremely remarkable irritation.

Comparative example 3 is different from example 1 in that the rhodiola rosea fermentation lysate is not added, and only the soybean lecithin and the like are used for coating the glabridin, so that the cytotoxicity of the glabridin is reduced to a certain extent (see comparative example 3 and comparative example 4), but the cytotoxicity is still significantly higher than that of example 1. Therefore, the combination of the rhodiola rosea fermentation lysate, the glabridin and the liposome containing soybean lecithin and the like can further remarkably improve the dispersion capacity of the glabridin in water and the stability of the system, reduce the content of the non-coated glabridin in the system, and further reduce the cytotoxicity of the system.

Comparative example 6 the amount of glabridin added was too high compared with examples 1 to 6, and the amount of glabridin coated in the final product was limited, and there was a large amount of free, uncoated glabridin in the system, resulting in an increase in toxicity to HaCat cells.

Comparative example 7 differs from example 1 only in that the rhodiola rosea fermentation lysate was replaced with an equal amount of rhodiola rosea fermentation product filtrate, and the product (80.52%) obtained in comparative example 7 was 10.24% higher in cell viability than in comparative example 3 (70.28%), but the product obtained in example 1 was lower in cytotoxicity, i.e. higher in viability (93.62% in example 1). It is presumed that the rhodiola rosea fermentation lysate used in example 1 contains more substances which are favorable for dispersing the glabridin and improving the stability of the system, so that the non-coated glabridin content in the system is reduced, and the cytotoxicity of the system to HaCat is reduced.

Effect example 6 measurement of melanogenesis inhibition ratio

Taking logarithmic growth phaseWell conditioned B16 cells were counted and seeded in 6-well plates at 37℃in 5% CO ₂ Culturing overnight in an environment incubator, and washing with PBS for 2 times when the cell fusion rate reaches more than 80%.

Experimental group: 2mL of the sample solution of the example or the sample solution of the comparative example was added (the products obtained in the above examples or comparative examples were diluted with serum-free DMEM to a volume percentage of 0.06% of the sample solution, respectively).

Blank group: only 2mL of serum-free medium was added.

After each group was added, the culture was carried out for 24 hours, the old medium was removed after the culture, washed with PBS, then 500. Mu.L of pancreatin was added to digest for 4 minutes, 1mL of serum-containing culture solution was added to stop the digestion, centrifugation was carried out at 1500rpm for 3 minutes, the supernatant was removed by suction, cell precipitation was left, 0.5mL of 1M sodium hydroxide solution containing 10% DMSO was added, ultrasonic dispersion was carried out for 3 minutes, water bath was carried out at 80℃for 30 minutes, shaking and mixing were carried out, centrifugation was carried out at 10000rpm for 2 minutes, 200. Mu.L of supernatant was sucked into 96 well plates, and absorbance was measured at 405 nm.

Melanin production inhibition = 1-experimental OD/blank OD x 100%, 5 samples were repeated for each component, and the results were averaged, as shown in table 8 and fig. 5 (in table 8 and fig. 5, "x" indicates a significant difference from example 1, P <0.05; "x" indicates an extremely significant difference from example 1, P < 0.01).

TABLE 8 melanin production inhibition test results

Numbering device	Melanin production inhibition rate
		Example 1	24.38％
Example 2	25.12％
		Example 3	21.89％
Example 4	19.78％
		Example 5	14.18％
Example 6	10.20％
		Comparative example 1	16.79％*
Comparative example 2	8.71％**
		Comparative example 3	7.96％**
Comparative example 4	22.64％
		Comparative example 5	10.82％*
Comparative example 6	19.78％
		Comparative example 7	19.40％*

According to the results, the products prepared in examples 1 to 6 of the present application have ideal melanin production inhibiting effects, and thus can be seen to have ideal whitening effects.

In example 1, the melanin production inhibition rate was significantly improved as compared with that in example 1. Therefore, the whitening effect of the liposome solution prepared by taking the yeast rhodiola rosea fermentation lysate as a raw material is better than that of the liposome solution prepared by taking the lactic acid bacteria rhodiola rosea fermentation lysate as a raw material.

According to the effect data of comparative example 2, comparative example 3 and example 1, the addition amount of the rhodiola rosea fermentation lysate in the application is relatively small, and the rhodiola rosea fermentation lysate has a certain whitening effect, but is relatively weak (see comparative example 2), but when the rhodiola rosea fermentation lysate is used in combination with glabridin, liposome including soybean lecithin and the like, the melanin production inhibition rate of the system can be remarkably improved, and the three components have synergistic promotion effects on the melanin production inhibition rate after being combined, namely, the melanin production inhibition rate (24.38%) of the product prepared in example 1 is 7.71% higher than the sum of the melanin production inhibition rates of the products prepared in comparative example 2 (8.71%) and comparative example 3 (7.96%).

As is clear from the effect data of comparative example 4, the aqueous solution of glabridin, which was co-dissolved in an organic solvent, had an ideal melanin production-inhibiting activity, indicating that glabridin had a indeed whitening effect, but it was found from the above analysis that the product obtained in comparative example 4 was large in cytotoxicity and irritation and unsuitable as a skin external preparation.

In the comparative example 3, the rhodiola rosea fermentation lysate is not added, and only liposome including soybean lecithin and other substances is used for wrapping the glabridin, so that although the solubility and the dispersibility of the glabridin an aqueous phase can be improved, the whitening activity of the glabridin is weakened to a certain extent, the wrapped glabridin is presumably not easy to release, and further the glabridin cannot play a role in a system.

Comparative example 6 is too high in the amount of glabridin as compared with examples 1 to 6, and the system is unstable, so that glabridin has poor dispersibility in the system, and the glabridin undergoes self-aggregation, and although the self-aggregated glabridin can enter cells to exert a certain whitening effect by phagocytosis of cells, the product prepared in comparative example 6 has high cytotoxicity and irritation, and is not suitable for being used as an external preparation for skin, as is known from the above analysis.

The melanin production inhibition rate of the product prepared in example 1 was significantly improved as compared to comparative example 7. The combination of the rhodiola rosea lysate and soybean lecithin and other substances is more beneficial to improving the solubility and dispersion stability of the glabridin in the system, and improves the bioavailability of the glabridin to a certain extent while reducing the irritation of the glabridin, does not influence the release of the glabridin, and further improves the whitening effect of the system.

Effect example 7

Skin surface gloss is reflected by direct reflection and scattering of light impinging on the skin surface. An LED at the tip of the probe produces a parallel beam of white light that passes through a planar mirror and is directed at the skin surface at an angle of 60 degrees, and a portion of the light is reflected directly at the same angle and then passes through a planar mirror to a receiving transducer. Another portion of the light is scattered by the skin surface and received by another sensor. The skin gloss test probe GL200 can thus test not only the light directly reflected by the skin in relation to the gloss, but also the light scattered by the skin. Parallel white light is produced by the LEDs at the top of the probe. Two independent sensors test the reflected light and scattered light, respectively, after passing through a plane mirror at the same angle of incidence. Based on the assumption that the incident light is scattered in the same way in all angular directions, the test of scattered light is performed at an angle of 0 ° of the incident light that is completely perpendicular to the skin surface.

The improvement ability of the essence prepared in the application example 1 on the skin glossiness is tested, and the method comprises the following steps:

selecting facial skin dry, lack of moisture, and test area moisture content Coreometer screen value<60au; facial skin barrier function is poor and test areasTEWL screening value at 12g/m ² h～30g/m ² h is between; facial skin darkness lacks gloss and test area gloss glossmeter screen value<9 au; 30 Chinese healthy male/female subjects with a certain degree of red acne marks on the face in the post-acne or recovery phase of the acne, and the age range is 18-40 years. Skin gloss was measured before and after the test product, for 14 days and 28 days, respectively, using the test product using the front and rear control method. The results of the evaluation of the products before and after use were compared by a statistical test method to determine whether there was a statistical difference, and the results are shown in Table 9 and FIG. 6.

TABLE 9 skin gloss test results

When the skin is smooth, has less wrinkles and is flat, the skin appears shiny; when the skin is rough and lacks oil, scattering of light is caused to a large extent, and the skin is tarnished. The skin gloss was improved by 11.37% and 18.86%, respectively, measured after 14 days and 28 days using the essence prepared in application example 1. And the skin reddening and other stimulation phenomena do not occur in the using process. Therefore, the essence prepared in application example 1 has ideal effect of improving skin glossiness and high use safety.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While the disclosure has been disclosed by the foregoing description of specific embodiments thereof, it will be understood that various modifications, improvements, or equivalents may be devised by those skilled in the art that will fall within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this disclosure.

Claims

1. A method for preparing a liposome solution, comprising the steps of:

(2) Mixing 1-4 parts of lecithin, 0.05-0.125 part of cholesterol, 0.3-0.8 part of polyglycerol emulsifier, 1-4.5 parts of polyalcohol and 0.075-0.5 part of glabridin with 1-4 parts of rhodiola rosea fermentation lysate prepared in the step (1) and 15-25 parts of water, homogenizing, and homogenizing under high pressure to prepare a liposome solution;

In the step (1), the preparation method of the red scenic spot water extract comprises the following steps: leaching dried rhodiola rosea and water for 1-3 hours at the temperature of 85-95 ℃, centrifuging, and collecting supernatant, wherein the mass ratio of the dried rhodiola rosea to the water is (1-5): 100; the saccharomycete is saccharomyces cerevisiae; the fermentation culture time is 24-72 h; the power of the ultrasonic wave is 65-130W;

in the step (2), the lecithin is at least one of soybean lecithin, egg yolk lecithin and phosphatidylcholine lecithin; the polyglycerol emulsifier is at least one of polyglycerol-10 stearate, polyglycerol-10 laurate, polyglycerol-10 myristate, polyglycerol-10 palmitate and polyglycerol-10 oleate; the polyalcohol is at least one of dipropylene glycol, glycerol, propylene glycol, butanediol and pentanediol; the high-pressure homogenizing pressure is 200-800 bar.

2. The method of preparing a liposome solution according to claim 1, wherein the method of preparing a liposome solution satisfies at least one of the following conditions:

in the step (1), the rhodiola rosea water extract also comprises a sterilization operation before use;

In the step (1), the number of the yeasts inoculated in the red scenic spot water extract per unit volume is 10 ⁵ ~10 ⁶ CFU/mL；

In the step (1), the temperature of fermentation culture is 25-35 ℃;

in the step (1), the ultrasonic time is 5-20 min.

3. The method of preparing a liposome solution according to claim 2, wherein the method of preparing a liposome solution satisfies at least one of the following conditions:

in the step (1), in the preparation process of the rhodiola rosea water extract, the mesh number of the rhodiola rosea is 10-100 meshes;

in the step (1), in the preparation process of the red scenic spot water extract, the leaching temperature is 90-95 ℃;

in the step (1), in the preparation process of the red scenic spot water extract, the leaching time is 1-2 hours;

in the step (1), in the preparation process of the red scenic spot water extract, the rotational speed of the centrifugation is 4000-5000 rpm;

in the step (1), during the preparation process of the red scenic spot water extract, the centrifugation time is 25-40 min;

in the step (1), in the preparation process of the red scenic spot water extract, the operation of centrifuging further comprises the operation of filtering the supernatant;

In the step (1), the saccharomycete is at least one of "saccharomyces cerevisiae with a preservation number of cic 1308" purchased from a China industry microbiological culture collection center "," saccharomyces cerevisiae with a preservation number of cic 1305 "purchased from a China industry microbiological culture collection center", "saccharomyces cerevisiae with a preservation number of cic 1002" and "saccharomyces cerevisiae with a preservation number of cic 1346" purchased from a China industry microbiological culture collection center;

in the step (1), the fermentation culture time is 48-72 h;

in the step (1), the temperature of fermentation culture is 25-28 ℃;

in the step (1), the ultrasonic time is 15-20 min.

4. The method for preparing a liposome solution according to any one of claims 1 to 3, wherein the method for preparing a liposome solution satisfies at least one of the following conditions:

in the step (2), the weight part of the cholesterol is 0.1-0.125 part;

in the step (2), the weight part of the polyglycerol emulsifier is 0.5-0.8 part;

in the step (2), the weight part of the polyol is 2.5-3.5 parts;

In the step (2), the glabridin is 0.075-0.25 parts by weight;

in the step (2), the rhodiola rosea fermentation lysate is 2-4 parts by weight;

in the step (2), the weight part of the water is 19-25 parts;

in the step (2), a preservative is further added in the mixing process;

in the step (2), the homogenizing temperature is 70-80 ℃;

in the step (2), the homogenizing time is 5-15 min;

in the step (2), the homogenization is performed in a homogenizer, and the rotation speed of the homogenizer is 5000-10000 rpm;

in the step (2), the high-pressure homogenization time is 5-10 min.

5. The method of claim 4, wherein the method of preparing a liposome solution satisfies at least one of the following conditions:

in the step (2), the phosphatidylcholine lecithin comprises dimyristoyl lecithin and/or dipalmitoyl phosphatidylcholine;

in the step (2), the glabridin is 0.125-0.25 part by weight;

in the step (2), the weight part of the preservative is 0.25-2 parts;

In the step (2), the preservative comprises an alcohol preservative and/or p-hydroxyacetophenone;

in the step (2), the homogenizing temperature is 75-80 ℃;

in the step (2), the homogenization is performed in a homogenizer, and the rotation speed of the homogenizer is 7000-8000 rpm;

in the step (2), the high-pressure homogenizing pressure is 600-800 bar.

6. A liposome solution prepared by the method for preparing a liposome solution according to any one of claims 1 to 5.

7. Use of the liposome solution of claim 6 directly as a product, as an additive or as a substrate in the preparation of a skin external agent.

8. A skin external preparation comprising the liposome solution according to claim 6.

9. The external skin preparation according to claim 8, wherein the external skin preparation satisfies at least one of the following conditions:

the skin external agent further comprises at least one of a humectant, an emollient, a preservative, a thickener, a pH adjustor, a chelating agent, and a solvent;

the external skin preparation is a facial mask, essence or toner;

the liposome solution accounts for 1-20% of the skin external agent by mass.

10. The external skin preparation according to claim 9, wherein the external skin preparation satisfies at least one of the following conditions:

the humectant comprises a polyhydric alcohol humectant and/or betaine;

the emollient comprises at least one of polydimethylsiloxane, caprylic/capric triglyceride, and glyceryl caprylate;

the preservative comprises phenoxyethanol;

the thickener comprises acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer;

the pH adjuster comprises arginine;

the chelating agent comprises disodium EDTA;

the solvent comprises deionized water;

the mass ratio of the humectant to the liposome solution is (4-7): 1, a step of;

the mass ratio of the emollient to the liposome solution is (0.5-1.5): 1, a step of;

the mass ratio of the preservative to the liposome solution is (0.1-0.2): 1, a step of;

the mass ratio of the thickener to the liposome solution is (0.05-0.15): 1, a step of;

the mass ratio of the pH regulator to the liposome solution is (0.05-0.15): 1, a step of;

the mass ratio of the chelating agent to the liposome solution is (0.02-0.05): 1, a step of;

the mass ratio of the solvent to the liposome solution is (35-45): 1.