Use of biotransformed bear bile powder in the manufacture of cosmetics and cosmetics

Abstract

The present invention provides a use of multi-functional, multi-passage biotransformed bear bile powder in the manufacture of cosmetics. [Solution] There is provided a use of biotransformed bear bile powder in the manufacture of cosmetics, characterized in that the biotransformed bear bile powder contains bile acids, the bile acids including tauroursodeoxycholic acid, taurochenodeoxycholic acid and cholic acid.

Description

[Technical field]

[0001] The present invention relates to the technical field of cosmetics, especially to the field of IPC classification code A61K8 / 98, specifically to the use of biotransformed bear bile powder in the production of cosmetics and the cosmetics. [Background technology]

[0002] Bear bile is a precious animal-derived traditional Chinese medicine with the effects of clearing heat, antispasmodic, improving eyesight, and toning the liver and gallbladder, and its medicinal use has a history of more than two thousand years. At present, the raw material of bear bile for medicinal or health products is extracted and separated from the bile of Asiatic black bears or brown bears, but the supply from the industry that raises bears to obtain bile is limited, and the development of non-essential bear bile products by companies is prohibited by the state, so the current production of bear bile cannot meet people's increasing health needs, and the progress of research into the use of bear bile is also greatly restricted.

[0003] Natural bear bile powder is expensive, so there are few reports in the prior art on the use of bear bile powder in cosmetics. Chinese patent application CN1190578A discloses that bear bile powder is added to cosmetics to emulsify and decompose the sebum of the human body. Chinese patent application CN104887601A discloses cosmetics containing bear bile powder, which is a mixture of bear bile powder and various oils and fats, and has the effect of moisturizing and beautifying, but it is not clear what effect bear bile powder has in it.

[0004] In recent years, scientists have focused on the research of artificial bear bile powder and developed a series of methods for producing artificial bear bile powder, but most of them are separated and purified by physical or chemical methods to obtain products similar to the components of natural bear bile powder. The artificial bear bile powder produced by these methods has problems such as complex components, long production cycle, low content of TUDCA (tauroursodeoxycholic acid), high toxicity and side effects, and cannot meet the requirements for use. The prior patent in which the inventor participated in the research and development, Chinese patent CN104382941B, discloses a method for producing artificial bear bile powder, and realizes the production of artificial bear bile powder from poultry and livestock bile powder, and the produced bear bile powder is similar to the components of natural bear bile powder. As artificial bear bile powder gradually enters the market, it provides space for research into the development of artificial bear bile powder for non-medicinal uses. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a multi-function, multi-passage biotransformed bear bile powder for use in making cosmetics.

[0006] Another object of the present invention is to provide a cosmetic product for the above-mentioned use. [Means for solving the problem]

[0007] In order to achieve the above object of the invention, the present invention uses the following technical means.

[0008] In the use of the biotransformed bear bile powder in the manufacture of cosmetics, the biotransformed bear bile powder contains bile acids, including tauroursodeoxycholic acid (TUDCA, CAS number 14605-22-2), taurochenodeoxycholic acid (TCDCA, CAS number 516-35-8) and cholic acid (CA, CAS number 81-25-4).

[0009] Preferably, the mass fraction of bile acids in the biotransformed bear bile powder is 60-75%, more preferably 63.77%-72.48%, and even more preferably 69.58%.

[0010] Preferably, the bile acids further comprise 7-keto-TLCA (7-ketokenodeoxycholic acid, CAS number 4651-67-6).

[0011] Preferably, the mass ratio of the tauroursodeoxycholic acid, taurochenodeoxycholic acid, and cholic acid is 1.1-1.5:1:0.3-0.5.

[0012] Preferably, the mass ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid is 1.12 to 1.37:1, more preferably 1.25:1.

[0013] Preferably, the 7-keto-TLCA accounts for 0.31-1.08% of the mass of the biotransformed bear bile powder.

[0014] Preferably, the biotransformed bear bile powder further comprises amino acids, proteins and trace elements.

[0015] Preferably, the amino acids account for 1.37-3.40% of the mass of the biotransformed bear bile powder.

[0016] Preferably, the amino acids include taurine, glycine, alanine, leucine, and valine.

[0017] Preferably, the amino acids, by mass percentage of the biotransformed bear bile powder, include 0.09-0.72% taurine, 0.33-1.90% glycine, 0.03-0.21% alanine, 0.01-0.68% leucine, 0.01-0.51% valine, 0.00-0.37% phenylalanine, 0.00-0.13% proline, 0.00-0.10% methionine, 0.00-0.01% threonine, and 0.00-0.20% serine.

[0018] Preferably, the protein accounts for 4.82-8.27% of the mass of the biotransformed bear bile powder.

[0019] The biotransformed bear bile powder selected in the present invention is rich in bile acids, and also contains a variety of amino acids and proteins, all of which are edible ingredients, are safe, and can be used in skin care products; at the same time, the biotransformed bear bile powder of the present invention is almost non-irritating to the skin, which is believed to be due to the low content of highly acidic ingredients such as 7-keto-TLCA. In addition, the biotransformed bear bile powder of the present invention has high solubility in water, with a solubility of about 17.4g / 100g at room temperature, and has no special odor, meeting the requirements for use in cosmetics.

[0020] Preferably, the bioconverted bear bile powder comprises: S1, dispersing poultry bile powder in glycine buffer, adding 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution, adjusting the pH, and carrying out the enzyme conversion reaction for 6 to 18 hours; S2: The product after the enzymatic conversion reaction in step S1 is rotary evaporated, washed, concentrated and dried to obtain bioconverted bear bile powder.

[0021] Preferably, the TCDCA content of the poultry bile powder is 70% or more.

[0022] Preferably, the poultry bile powder is produced by the following steps M1 to M3.

[0023] In M1, the bile of poultry is cut and the bile is collected, condensed for 10 to 30 h at 0 to 4 °C, and filtered through a filter membrane with a pore size of 0.25 to 0.45 μm to remove the condensed oil and fat, and the liquid phase is retained.

[0024] In M2, the liquid phase is concentrated to 1 / 3 to 1 / 2 volume to obtain extract I, to which 2 to 10 times the volume of water is added, and the extract is stirred uniformly at 0 to 4°C, allowed to stand for 10 to 30 hours, and then filtered through a filter membrane with a pore size of 0.25 to 0.45 μm.

[0025] In M3, the filtrate is concentrated until the volume is the same as that of extract I to obtain extract II, to which 2 to 10 times the volume of 90 to 95% aqueous ethanol solution is added, and the mixture is stirred uniformly at 0 to 4°C, and then allowed to stand for 10 to 30 hours. The mixture is then filtered through a filter membrane with a pore size of 0.25 to 0.45 μm, concentrated and dried to obtain poultry bile powder.

[0026] Preferably, the stirring rotation speed is 30 rpm to 80 rpm.

[0027] Preferably, the poultry bile powder comprises one or more of chicken bile powder, duck bile powder, goose bile powder produced by the above method from chicken bile, duck bile, and goose bile, respectively.

[0028] Preferably, the 7α-hydroxysteroid dehydrogenase conversion liquid is obtained by producing enzyme cell A with reference to the method described in the prior patent CN111407780A, and dispersing the enzyme cell A in 50 mL of glycine buffer per 10 g.

[0029] Preferably, the 7β-hydroxysteroid dehydrogenase conversion liquid is obtained by producing enzyme bacteria B with reference to the method described in the prior patent CN111407780A, and dispersing the enzyme bacteria B in 50 mL of glycine buffer per 10 g.

[0030] Preferably, in the method for producing bioconverted bear bile powder, the mass ratio of enzyme bacteria A to enzyme bacteria B is 1:2-10, and more preferably 1:6-10.

[0031] Preferably, the 7α-hydroxysteroid dehydrogenase conversion solution occupies 3-5% of the volume of the reaction solution for the enzymatic conversion reaction in step S1, more preferably 4%; Preferably, the 7β-hydroxysteroid dehydrogenase conversion solution occupies 30-50% by volume of the reaction solution for the enzymatic conversion reaction in step S1, and more preferably occupies 40%.

[0032] The reaction solution for the enzyme conversion reaction is a mixture of poultry bile powder, glycine buffer, 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution.

[0033] Preferably, the concentration of the glycine buffer is 80 to 120 mM, more preferably 100 mM.

[0034] Preferably, the concentration of the poultry bile powder in the reaction solution for the enzymatic conversion reaction in step S1 is 50 to 250 g / L.

[0035] Preferably, the pH is from 6 to 9, and more preferably, 7.5.

[0036] The temperature of the enzymatic conversion reaction is preferably 20°C to 30°C, and more preferably 25°C.

[0037] Preferably, the time for the enzymatic conversion reaction is 14 to 16 hours.

[0038] Preferably, the cosmetic product is an antioxidant cosmetic product.

[0039] Preferably, the cosmetic product has the effect of removing free radicals.

[0040] Preferably, the cosmetic product is a sebum-controlling, anti-acne cosmetic product, Preferably, the cosmetic product is capable of suppressing sebum secretion.

[0041] Preferably, the cosmetic product has an inhibitory effect on sebaceous gland cells.

[0042] Preferably, the cosmetic product is a mild cosmetic product.

[0043] Preferably, the cosmetic product has the effect of suppressing the secretion of inflammatory mediators by macrophages.

[0044] Preferably, the cosmetic product has an inhibitory effect on mast cell degranulation.

[0045] Preferably, the cosmetic product is a protective cosmetic product.

[0046] Preferably, the cosmetic product has a protective effect against damaged cells.

[0047] In order to further expand the application field of biotransformed bear bile powder and provide new active raw materials in the cosmetics field, the inventors have found in their research that biotransformed bear bile powder produced by a specific method has skin care activity in many aspects, and shows effective antioxidant, sebum control, irritation relief and protection effects. Biotransformed bear bile powder can remove free radicals and shows antioxidant activity, thereby playing the role of anti-aging and anti-photoaging. Biotransformed bear bile powder also shows an activity inhibitory effect on sebaceous gland cells SZ95, reducing sebum secretion and avoiding problems such as acne caused by excess sebum. In particular, biotransformed bear bile powder only inhibits the activity of SZ95, and has almost no cytotoxicity and does not affect the normal function of the skin. At the same time, after administration of biotransformed bear bile powder, the amount of inflammatory mediator TNF-α secreted by macrophages was significantly reduced, and the degranulation rate of mast cells was also significantly reduced, showing excellent anti-inflammatory irritation relief activity. In addition, for damaged tissue cells, biotransformed bear bile powder significantly increases the number of viable cells, reduces the loosening of the stratum corneum, and shows a significant protective effect on skin that has lost tissue viability after stimulation.

[0048] Another aspect of the present invention provides a cosmetic for the above-mentioned use, the cosmetic being a lotion, milky lotion, face cream, eye cream, essence, pack, facial cleanser, shampoo, body soap, toilet soap, sunscreen cream or hand cream, the cosmetic further comprising additives acceptable in the cosmetic field in addition to biotransformed bear bile powder.

[0049] Preferably, the cosmetic product is a face cream, and the ingredients of the face cream include stearic acid ester, stearyl alcohol, glycerin, water, thickener, preservative and biotransformed bear bile powder; Preferably, the bio-transformed bear bile powder accounts for 2-10% of the mass of the face cream; For example, the bio-transformed bear bile powder comprises 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the mass of the face cream. Effect of the Invention

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The present invention takes the lead in using biotransformed bear bile powder in cosmetics, further opening up the application field of artificial bear bile powder, and has great industrial value.

[0052] 2. The biotransformed bear bile powder used in the present invention is low-cost, made from edible raw materials, has safe and environmentally friendly ingredients, caters to the market hotspot of "medicinal cosmetics", and meets consumer preferences.

[0053] 3. The present invention pioneered the discovery that the artificial bear bile powder produced by biotransformation method has antioxidant activity, can effectively eliminate free radicals, and has certain anti-aging and anti-photoaging effects.

[0054] 4. The present invention pioneered the discovery that biotransformed bear bile powder can inhibit sebum secretion from sebaceous gland cells, exert the effect of sebum control, and help prevent the occurrence of acne.

[0055] 5. The present invention pioneered the discovery that biotransformed bear bile powder has anti-inflammatory and irritation-relieving effects, and can inhibit the occurrence of inflammation through multiple pathways and exert irritation-relieving effects.

[0056] 6. The present invention creatively discovers that biotransformed bear bile powder has a repairing effect, and has a significant effect on repairing and revitalizing damaged cells in skin tissue. [Brief description of the drawings]

[0057] [Figure 1] This is a relationship graph between the ABTS free radical scavenging rate and concentration of biotransformed bear bile powder. [Diagram 2] FIG. 11 is a graph showing the relationship between the concentration of biotransformed bear bile powder and the cell viability of SZ95 cells. [Diagram 3] 13 is a Nile Red stained fluorescence microscope photograph of lipid droplets in a sebum control test of biotransformed bear bile powder. [Figure 4] This is a relationship graph between lipid droplet content and concentration in a sebum control test of biotransformed bear bile powder. [Diagram 5] FIG. 11 is a graph showing the relationship between the concentration of biotransformed bear bile powder and the cell viability of macrophages RAW264.7. [Figure 6] FIG. 13 is a graph showing the relationship between TNF-α content and the concentration of bioconverted bear bile powder in the measurement of inflammatory factors. [Figure 7] FIG. 13 is a graph showing the relationship between PGE2 content and the concentration of bioconverted bear bile powder in the measurement of inflammation factors. [Figure 8] FIG. 1 is a graph showing the relationship between the concentration of biotransformed bear bile powder and the cell viability of mouse mastocytoma cell P815. [Figure 9] 1 shows micrographs of the state of cell degranulation in each group in the measurement of mast cell degranulation. [Figure 10] FIG. 1 is a graph showing the relationship between the degranulation rate in a mast cell degranulation assay and the concentration of bioconverted bear bile powder. [Figure 11] 13 is a micrograph of a skin model after administration for histomorphometric analysis. [Figure 12] 1 is a trend graph of relative tissue viability in tissue viability measurements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Example 1 This embodiment provides the use of bio-transformed bear bile powder in the preparation of cosmetics, and the main components and contents of the bio-transformed bear bile powder are shown in Table 1.

[0059] Table 1 [Table 1]

[0060] The biotransformed bear bile powder of this embodiment is produced according to the following methods S1 and S2.

[0061] In S1, 250 g of chicken bile powder was dispersed in 300 mL of 100 mM glycine buffer, 40 mL of 7α-hydroxysteroid dehydrogenase conversion solution and 400 mL of 7β-hydroxysteroid dehydrogenase conversion solution were added, 100 mM glycine buffer was added until the total volume reached 1 L, the pH was adjusted to 7.5 with 5 M NaOH aqueous solution, and the enzyme conversion reaction was carried out at 25°C for 16 hours.

[0062] In step S2, the product of the enzymatic conversion reaction in step S1 was rotary evaporated at 65°C until half the volume remained, then washed with 10 times the volume of 95% ethanol aqueous solution, and centrifuged to remove the precipitate. The supernatant was evaporated at 75°C to the volume before washing, and vacuum dried at 85°C to obtain bioconverted bear bile powder.

[0063] Here, chicken bile powder is produced by the following methods M1 to M3.

[0064] In M1, chicken bile was cut to collect the bile, which was then condensed for 24 h at 2°C, and filtered through a membrane filter with a pore size of 0.45 μm to remove the condensed oil and fat, and the liquid phase was retained.

[0065] In M2, the liquid phase is concentrated under reduced pressure at 65°C in an external circulation evaporator until it becomes 1 / 2 of its volume, to obtain extract I, which is added with 10 times the volume of water, stirred uniformly at 60 rpm at 2°C, allowed to stand for 24 hours, and then filtered through a filter membrane with a pore size of 0.45 μm.

[0066] In M3, the filtrate is concentrated under reduced pressure in an external circulation evaporator at 65°C until it becomes the same volume as extract I, to obtain extract II, to which 10 times the volume of 95% ethanol aqueous solution is added, and the mixture is stirred uniformly at 2°C, allowed to stand for 24 hours, filtered through a 0.45 μm pore membrane, further concentrated under reduced pressure in an external circulation evaporator at 65°C, and dried at 80°C in a vacuum belt dryer to obtain chicken bile powder.

[0067] The 7α-hydroxysteroid dehydrogenase conversion solutions and 7β-hydroxysteroid dehydrogenase conversion solutions used in this example are from the preferred sources described herein.

[0068] The cosmetic product is an antioxidant cosmetic product.

[0069] This embodiment further provides a cosmetic product according to the above use, the cosmetic product being a face cream, the ingredients of the face cream including, by weight percentage of the face cream, 10% white oil, 1% PEG-60, 2.5% propylheptyl caprylate, 4% cetearyl alcohol, 3% glycerin, 1% xanthan gum, 1.5% undecane (and) tridecane, 0.05% EDTA, 0.5% phenoxyethanol, 3% biotransformed bear bile powder, and the remainder deionized water.

[0070] Example 2 This example provides the use of biotransformed bear bile powder in the manufacture of cosmetics and the cosmetics in said use, and the embodiment is the same as Example 1, except that the cosmetic is a sebum-controlling and anti-acne cosmetic.

[0071] Example 3 This example provides the use of biotransformed bear bile powder in the manufacture of cosmetics and the cosmetics in said use, and the embodiment is the same as that of Example 1, except that the cosmetics are mild cosmetics.

[0072] Example 4 This example provides the use of biotransformed bear bile powder in making cosmetics and the cosmetics in said use, and the embodiment is the same as that of Example 1, except that the cosmetics are repair cosmetics.

[0073] Performance Testing 1. Antioxidant performance test ABTS free radical scavenging ability test: Using PBS buffer solution of pH 7.4, the biotransformed bear bile powder in Example 1 was prepared into sample solutions with concentrations (m / V) of 1.56%, 3.13%, 6.25%, 12.5%, and 25%.

[0074] The reaction groups C1, C2, T1, and T2 were prepared in the following order with the following system mixing ratios, and after incubation at 25°C for 5 min, the absorbance OD value at 734 nm was measured to calculate the ABTS free radical scavenging rate.

[0075] [Table 2]

[0076] Here, the ABTS working solution was derived from the ABTS method of the Biyuntian total antioxidant capacity measurement kit, the test solutions were the bioconverted bear bile powder sample solutions of each concentration prepared above, the sample solvent was PBS buffer solution at pH 7.4, and for each test solution, three replicate wells and one background control well were set up.

[0077] The calculation method for the ABTS free radical scavenging rate is as follows:

number

[0078] The results of ABTS free radical scavenging rate are shown in Table 2 and FIG. 1, where BC is the blank control.

[0079] Table 2 [Table 3]

[0080] In Figure 1, statistical analysis was performed using the t-test method to determine the significance of the comparison between the sample group and the BC group. * and p-value < 0.05 is indicated. * and p-value<0.01 is indicated. ** As shown in.

[0081] 2. Sebum control test of sebaceous gland cells SZ95 cell inhibition test: Reagents: DMEM medium (Gibco), fetal bovine serum (Gibco), PBS (Sigma), MTT (Sigma), DMSO (Sigma), trypsin (Gibco). Sample: Biotransformed bear bile powder from Example 2.

[0082] 2.1. Cytotoxicity test 1) Cell inoculation: 1×10 4 SZ95 cells were seeded into a 96-well plate at a seeding density of cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0083] 2) Grouping of the test: The test included a blank group, a control group, a positive control group, and a sample group. In the sample group, eight step concentrations were set for each sample, and three duplicate wells were set for each concentration. The eight concentrations were as follows, and the solvent was PBS buffer at pH 7.4.

[0084] [Table 4]

[0085] 3) Administration: Administration was performed when the ratio of the cells occupying the bottom area in the 96-well plate reached 40% to 60%. 200 μL of culture medium containing 10% PBS was added to each well of the control group, 200 μL of culture medium containing 10% DMSO was added to each well of the positive control group, 200 μL of culture medium containing the corresponding concentration of sample was added to each well of the sample group, and no cells were inoculated in the blank group, only 200 μL of cell culture medium was added. After administration was completed, the 96-well plate was placed in an incubator (37°C, 5% CO2) and cultured.

[0086] 4) Measurement: After incubating the cells for 24 hours, the supernatant was discarded, a medium containing 0.5 mg / mL of MTT was added, and the cells were incubated at 37°C for 2 hours away from light. After the incubation was completed, the supernatant was discarded, 100 μL of DMSO was added to each well, the OD value was read at 490 nm, and the relative cell viability was calculated using the following formula.

[0087]

number

[0088] The measurement results are shown in Table 3 and FIG.

[0089] Table 3 [Table 5]

[0090] 2.2. Sebum control test 1) Cell inoculation: 1×10 5 SZ95 cells were seeded into a 24-well plate at a seeding density of cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0091] 2) Working solutions for each test group were prepared as follows:

[0092] [Table 6]

[0093] 3) Administration: According to the above test design, when the ratio of cells occupying the bottom area in the 24-well plate reached 40% to 60%, each group was administered with a dose of 1.0 mL, and three duplicate wells were set up for each group, followed by culturing in an incubator (37°C, 5% CO2) for 24 h.

[0094] 4) The medium was discarded, the cells were washed with PBS, fixed, and then stained with Nile Red for 15 minutes. After rinsing, the cells were photographed under a fluorescent microscope. The photographs are shown in Figure 3.

[0095] 5) Result analysis: Fluorescence intensity was quantitatively analyzed using Image Pro Plus software, and the lipid droplet content is shown in Table 4 and Figure 4.

[0096] Table 4 [Table 7]

[0097] Statistical analysis was performed using the t-test method. For comparison between the NC group and the BC group, significance was indicated with #, p-value<0.05 was indicated with #, and p-value<0.01 was indicated with ##. For comparison between the sample group and the NC group, significance was indicated with *, p-value<0.05 was indicated with *, and p-value<0.01 was indicated with **.

[0098] 3. Irritation relief test 3.1. Macrophage - Measurement of inflammatory factors Measurement cells: macrophage RAW264.7.

[0099] Main reagents: high glucose DMEM culture medium (VivaCell), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), DMSO (Kokuyo), trypsin (Gibco), LPS (Sigma), and dexamethasone (Sigma).

[0100] 3.1.1 Cytotoxicity test Referring to the method of 2.1, the cells were replaced with macrophages RAW264.7 to perform the MTT toxicity test, and the test results are shown in Table 5 and FIG.

[0101] Table 5 [Table 8]

[0102] 3.1.2, Inflammatory factor measurement 1) 1×10 5 RAW264.7 cells were seeded into a 24-well plate at a seeding density of cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0103] 2) Working solutions for each test group were prepared as follows:

[0104] [Table 9]

[0105] 3) Administration: According to the above test design, when the ratio of cells occupying the bottom area in the 24-well plate reached 40% to 60%, each group was administered with a dose of 1.0 mL, and three duplicate wells were set up for each group, followed by culturing in an incubator (37°C, 5% CO2) for 24 h.

[0106] 4) LPS stimulation: According to the test design, 100 μL of 10× LPS working solution was added to each well plate administered, and the well plate was shaken from side to side to ensure that the drug in the well plate was evenly mixed, and the final LPS concentration was 1 μg / mL. Then, the well plate was incubated in an incubator (37°C, 5% CO2) for 24 h.

[0107] 5) Sample collection: After incubation, the cell culture supernatant was collected in an EP tube, and the sample was then frozen and stored in a refrigerator at -80°C.

[0108] 6) Measurement of TNF-α content: Measurement was performed according to the instructions for the Mouse TNF-α ELISA kit.

[0109] Measurement of PGE2 content: The measurement was performed according to the instructions for the Prostaglandin E2 ELISA kit.

[0110] The results of the measurement of the TNF-α content are shown in Table 6 and FIG. 6, and the results of the measurement of the PGE2 content are shown in Table 7 and FIG.

[0111] Table 6 [Table 10] Table 7 [Table 11] Here, the definitions of # and * are the same as above.

[0112] 3.2 Measurement of mast cell degranulation Test cells: Mouse mast cell tumor cells P815 were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0113] Main reagents: high glucose DMEM culture medium (VivaCell), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), DMSO (Kokuyo), trypsin (Gibco), LPS (Sigma), and CCK-8 (Biyunten).

[0114] 3.2.1. Cytotoxicity test Referring to the method of 2.1, the cells were replaced with mouse mastocytoma cells P815, and the MTT toxicity test was carried out at a measurement wavelength of 450 nm. The test results are shown in Table 8 and FIG.

[0115] Table 8 [Table 12]

[0116] 3.2.2, Degranulation rate measurement method 1) 1×10 5 P815 cells were seeded into a 24-well plate at a seeding density of 1 cell / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0117] 2) Working solutions for each test group were prepared as follows:

[0118] [Table 13]

[0119] 3) Administration: According to the above test design, when the ratio of cells occupying the bottom area in the 24-well plate reached 40% to 50%, each group was administered with a dose of 1.0 mL, and three duplicate wells were set up for each group, followed by incubation in an incubator (37°C, 5% CO2) for 2 h.

[0120] 4) C48 / 80 stimulation: After incubating the samples for 2 hours, discard the waste liquid, and based on the test design, add 1mL of serum-free high glucose DMEM culture medium to the blank control group, add 1mL of serum-free high glucose DMEM culture medium containing C48 / 80 to the negative control group, add 1mL of serum-free high glucose DMEM culture medium containing C48 / 80 and sodium cromoglycate to the positive control group, add 1mL of serum-free high glucose DMEM culture medium containing C48 / 80 and a sample of the corresponding concentration to the sample group, and stimulate with C48 / 80 for 45 minutes after administration, and terminate the reaction in an ice bath.

[0121] 5) Observation of cell morphology: After the end of the culture, the degranulation status of the cells in each group was observed and photographed using an inverted microscope, as shown in Figure 9. The photographs were statistically analyzed using IPP software, and the cell degranulation rate was calculated using the following formula.

[0122]

number

[0123] The calculation results are shown in Table 9 and FIG.

[0124] Table 9 [Table 14] Here, the definitions of # and * are the same as above.

[0125] 4. Repair measurement Measurement model: Epidermal skin model (Skinovo (登録商標) -Epi).

[0126] Main Reagents: Maintenance culture medium (Skinovo (登録商標) ), PBS (Gibco), isopropanol (Kokuyaku), and 4% paraformaldehyde (Sigma).

[0127] The study design is as follows:

[0128] [Table 15]

[0129] Preparation of working stock solution 1) Preparation of 0.4% SLS stock solution: 0.0080 g of SLS was weighed out and dissolved in 2 mL of PBS solution, filtered through 0.22 μm, and a 0.4% SLS stock solution was prepared and ready for use.

[0130] 2) Preparation of 0.2% SLS working solution: 0.5 mL of 0.4% SLS solution was aspirated and added to 0.5 mL of PBS to prepare a 0.2% SLS working solution.

[0131] 3) Preparation of positive control 1 (dexamethasone) working solution: 100 mg of dried dexamethasone was dissolved in 1 mL of DMSO to prepare a stock solution with a concentration of 100 mg / mL. 6 μL of the 100 mg / mL stock solution was aspirated and dissolved in 6 mL of model culture medium to prepare a 0.01% dexamethasone working solution and ready for use.

[0132] 4) Preparation of working solution of positive control 2 (WY14643): 10 mg of WY14643 powder was weighed and dissolved in 1 mL of DMSO to prepare a 30 mM WY14643 stock solution. 10 μL of WY14643 stock solution (30 mM) was added to 6 mL of model culture medium to prepare a 50 μM working solution, ready for use.

[0133] Model administration: 1) Based on the above test design, the models were transferred to 24-well plates (0.9 mL of the model culture medium of the corresponding group was added in advance), and the 24-well plates were numbered according to the measurement group.

[0134] 2) 50μL of the prepared working solution for each group was applied to the model surface. 0.2% SLS solution was added to the model surface for the NC and PC groups, and the corresponding concentration of working solution was added to the sample group. To ensure that the sample was evenly distributed on the model surface, nylon film was sandwiched between sesame seeds and placed on the surface of each model. The models were then incubated in an incubator (37℃, 5% CO2) for 24 hours.

[0135] 3) After the incubation was completed, the test substance remaining on the model surface was washed off with a washing bottle containing sterile PBS solution, and any liquid remaining inside and outside the model was lightly wiped off with a sterile cotton swab.

[0136] 4.1, Histomorphometry After administration, the model was fixed with 4% paraformaldehyde and fixed for 24 hours. After that, the model was circumcised, stained with H&E, and photographed under a microscope for observation. The images were collected as shown in FIG. 11.

[0137] The histomorphometric micrographs showed that compared with the BC group, the stratum corneum of the epidermis model in the NC group was loosened and thickened, the viable cell layer was damaged, the number of viable cell layers was reduced, and vacuoles appeared, indicating that the SLS stimulation conditions were effective.

[0138] Compared with the NC group, the boundaries of the four-layer structure of the model in the PC (WY14643) group were clear, the cells in the viable cell layer were compactly arranged, the loosening phenomenon of the stratum corneum was significantly improved, and the number of viable cells increased, indicating that the positive control measurement in this study was valid.

[0139] Compared with the NC group, at a concentration of 3% of the sample biotransformed bear bile powder, the loosening phenomenon of the stratum corneum was significantly improved and the viable cells were significantly increased.

[0140] 4.2, Tissue viability measurement 1) MTT incubation: After washing the models after administration, prepare 24-well plates according to the number of models, label them accordingly, add 1 mL of MTT working solution (1 mg / mL) to each well, and place the 24-well plates containing the models in an incubator (37°C, 5% CO2) and incubate for 1 h.

[0141] 2) Isopropanol extraction: After MTT incubation, the outer surface of the model was washed with PBS, wiped dry with a sterile cotton swab, transferred to a new 24-well plate, labeled, 2 mL of isopropanol was added to the inner surface of the model, the 24-well plate was sealed with sealing film, and shaken on a microplate shaker for 2 h.

[0142] 3) Reading: The model was punctured with a pipette tip, and the isopropanol leachate was allowed to flow out from within the model into a 24-well plate. The punctured model was discarded, and the isopropanol leachate in each well was pipetted three times to thoroughly mix evenly. After mixing evenly, 200 μL of isopropanol leachate was aspirated from each well and added to the corresponding wells of a 96-well plate, which were then labeled. The optical density (OD) value was read at a wavelength of 570 nm using a microplate reader.

[0143] 4) Calculation: The relative tissue viability of each test group was calculated according to the following formula, and the results were recorded in Table 10, and the trend graph is shown in Figure 12.

[0144]

number

[0145] Table 10 [Table 16] Here, the definitions of # and * are the same as above.

[0146] 5. Toxicological testing According to the test and evaluation methods in Chapter 6 of the "Cosmetics Safety Technical Standards" (2015 edition), the biotransformed bear bile powder in Example 1 of the present invention was subjected to acute oral toxicity test, acute dermal toxicity test, acute eye irritation test, continuous skin irritation test, skin sensitization test, skin photosensitization test, skin phototoxicity test, skin photo test, and in vitro mammalian cell chromosomal aberration test. The toxicological test results are as follows:

[0147] 5.1. Acute oral toxicity test: The dermal LD50 of biotransformed bear bile powder in SD rats was >5000mg / kg body weight, and it was practically non-toxic. The body weights and mortality of the test animals (10 SPF grade SD rats) are shown in Table 11.

[0148] Table 11 [Table 17]

[0149] 5.2. Acute dermal toxicity test: The dermal LD50 of biotransformed bear bile powder in SD rats was ≧2180 mg / kg body weight, indicating slight toxicity. The body weights and deaths of the test animals (10 SPF grade SD rats) are shown in Table 12.

[0150] Table 12 [Table 18]

[0151] 5.3. Acute eye irritation (no wash) study: Biotransformed bear bile powder was found to be mildly irritating, with eye irritation response scores in three New Zealand rabbits shown in Table 13. In this study, a single dose of biotransformed bear bile powder was administered to each individual at a dose of 0.1 g.

[0152] Table 13 [Table 19]

[0153] 5.4. Continuous Skin Irritation Test: Biotransformed bear bile powder was found to have no irritating properties, with skin irritation response scores in four New Zealand white rabbits as shown in Table 14. In this test, the single dose of biotransformed bear bile powder administered to each individual was 0.5 g.

[0154] Table 14 [Table 20]

[0155] 5.5. Skin sensitization test (BT method): In the skin sensitization test of biotransformed bear bile powder in white guinea pigs, no skin sensitization was observed, and the skin reaction intensity of the white guinea pigs is as shown in Table 15, among which, the negative control was administered distilled water at the time of sensitization induction and 0.2g of biotransformed bear bile powder was applied at the time of sensitization induction, the positive control was applied with a 0.8% mass concentration of 2,4-dinitrochloropropiophenone in acetone solution at the time of sensitization induction and a 0.2% mass concentration of 2,4-dinitrochloropropiophenone in acetone solution at the time of sensitization induction, and the test substance group was applied with 0.2g of biotransformed bear bile powder at the time of sensitization induction and 0.2g of biotransformed bear bile powder at the time of sensitization induction.

[0156] Table 15 [Table 21]

[0157] 5.6. Skin phototoxicity test: Biotransformed bear bile powder showed no skin phototoxicity in a skin phototoxicity test using adult white guinea pigs, and the skin reaction scores of the guinea pigs are shown in Table 16. In this test, the dose of biotransformed bear bile powder administered to a single area of ​​a single individual was 0.2 g.

[0158] Table 16 [Table 22]

[0159] The numbers 1, 2, 3, and 4 in the skin reaction score title headers represent the different test treatments on four depilated sections of the same animal: 1 = biotransformed bear bile powder and no irradiation, 2 = biotransformed bear bile powder and irradiation, 3 = no biotransformed bear bile powder and no irradiation, and 4 = no biotransformed bear bile powder and irradiation.

[0160] 5.7. Skin photosensitization test Biotransformed bear bile powder showed no skin photosensitization in the skin photosensitization test in white guinea pigs, and the skin reaction scores in the light induction phase of white guinea pigs are shown in Table 17. In this test, 0.1 g of biotransformed bear bile powder was moistened with saline and administered to a single area of ​​a single individual in the light induction phase, and 0.02 g of biotransformed bear bile powder was moistened with saline and administered to a single area of ​​a single individual in the light excitation phase.

[0161] Table 17 [Table 23]

[0162] The skin reaction scores of 1, 2, 3 and 4 in the light induction stage correspond to the four areas where the test substance is applied and ultraviolet light is irradiated in the light induction stage, as shown in Figure 2 in Appendix 2 of Notice No. 136 of the State Food and Drug Administration of 2017 on the "Technical Specifications for Cosmetics Safety" (2015 edition).

[0163] 5.8. In vitro mammalian cell chromosomal aberration test: Biotransformed bear bile powder has no mutagenicity, and the chromosomal aberration rate is shown in Table 18. In this test, the cell line, metabolite activation system (liver microsomal enzyme mixture-S9 mixture) and positive samples were all derived from in vitro chromosomal aberration reagent bottles, the supplier was Beijing Huizhi Taikang Co., Ltd., and the reagent bottle lot number was 0221011 / 0221012.

[0164] Table 18 [Table 24] Notes: χ 2 Statistical analysis was performed using the test, and the results were compared with the negative control group. * P<0.05.

Claims

1. 1. Use of biotransformed bear bile powder in the manufacture of a sebum control, anti-acne cosmetic product, comprising: The biotransformed bear bile powder comprises bile acids, the bile acids comprising tauroursodeoxycholic acid, taurochenodeoxycholic acid and cholic acid; The bioconverted bear bile powder is S1: Dispersing chicken bile powder in glycine buffer, adding 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution, adjusting the pH to 7.5, and carrying out the enzyme conversion reaction at 25°C for 16 hours; S2. Rotary evaporating, washing, concentrating and drying the product after the enzymatic conversion reaction of step S1 to obtain bioconverted bear bile powder.

23. The method of claim 22,

2. The mass fraction of bile acids in the biotransformed bear bile powder is 60-75%; 2. The use of the biotransformed bear bile powder according to claim 1 in the manufacture of cosmetics for controlling sebum and preventing acne.

3. The mass ratio of the tauroursodeoxycholic acid, taurochenodeoxycholic acid, and cholic acid is 1.1 to 1.5:1:0.3 to 0.

5.

2. The use of the biotransformed bear bile powder according to claim 1 in the manufacture of cosmetics for controlling sebum and preventing acne.

4. The biotransformed bear bile powder further contains amino acids, proteins and trace elements; 2. The use of the biotransformed bear bile powder according to claim 1 in the manufacture of cosmetics for controlling sebum and preventing acne.

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