Application of biotransformation bear gall powder in preparation of cosmetics and cosmetics

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to application of biotransformation bear gall powder in preparation of cosmetics and the cosmetics. According to the invention, the biotransformation bear gall powder is creatively applied to cosmetics, and the artificial bear gall powder prepared by a biotransformation method has antioxidant activity and plays a certain role in resisting aging and light aging; sebum secretion of sebaceous gland cells can be inhibited, and acne can be prevented; meanwhile, the anti-inflammatory and soothing effects are achieved, inflammation can be inhibited from multiple channels, and the soothing effect is achieved; in addition, the selected biotransformation bear gall powder has a repairing effect, and has an obvious effect on repairing and activating damaged cells of skin tissues. The application field of the artificial bear gall powder is further expanded, and the selected biotransformation bear gall powder is low in cost, is selected from edible raw materials, is safe and environment-friendly in components, caters to the market hot spot of cosmeceutical, conforms to the preference of consumers and has great industrialization value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular to the field under IPC classification number A61K8 / 98, and more specifically to an application of biotransformed bear bile powder in the preparation of cosmetics and the cosmetics. Background Art

[0002] Bear bile is a precious animal-derived Chinese medicinal material with the effects of clearing away heat, relieving spasms, improving eyesight, protecting the liver and gallbladder. It has been used as medicine for more than 2,000 years. The bear bile raw materials currently used for medicine or health products are extracted and separated from the bile of black bears or brown bears, but the supply of bear bile is limited, and the state prohibits companies from developing non-essential bear bile products. Therefore, the current bear bile production cannot meet people's growing health needs, and it also greatly limits the progress of bear bile application research.

[0003] Due to the high cost of natural bear bile powder, there are few reports on the application of bear bile powder in cosmetics in the prior art. Patent CN 1190578A discloses that adding bear bile powder to cosmetics has the effect of emulsifying and degrading human sebum. Patent CN 104887601A discloses a cosmetic containing bear bile powder, which is a mixture of bear bile powder and various oils to achieve moisturizing and beauty effects, but it is not clear what role bear bile powder plays in it.

[0004] In recent years, scientists have focused on the research of artificial bear bile powder and developed a series of methods for manufacturing artificial bear bile powder. Most of them are separated and purified by physical or chemical methods to obtain products with similar components to natural bear bile powder. However, the artificial bear bile powder prepared by these methods has complex ingredients, long manufacturing cycle, low TUDCA (tauroursodeoxycholic acid) content, high toxicity and side effects, and other problems, which cannot meet the use requirements. The prior patent CN 104382941B developed by the inventors discloses a method for preparing artificial bear bile powder, which realizes the preparation of artificial bear bile powder from poultry and livestock bile powder. The prepared bear bile powder is similar to the components of natural bear bile powder. As artificial bear bile powder gradually enters the market, it also provides space for the application expansion research of artificial bear bile powder beyond medicinal use. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a multifunctional, multi-channel biotransformation bear bile powder for use in the preparation of cosmetics.

[0006] The present invention also aims to provide a cosmetic for the above application.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] The invention discloses an application of biotransformed bear bile powder in preparing cosmetics. The biotransformed bear bile powder contains bile acid; the bile acid includes tauroursodeoxycholic acid (TUDCA, CAS No. 14605-22-2), taurochenodeoxycholic acid (TCDCA, CAS No. 516-35-8) and cholic acid (CA, CAS No. 81-25-4).

[0009] Preferably, the mass fraction of bile acid in the bioconverted bear bile powder is 60-75%; further preferably, it is 63.77%-72.48%; more preferably, it is 69.58%.

[0010] Preferably, the bile acid further includes 7-keto-TLCA (7-ketotaurolithocholic acid, CAS No. 4651-67-6).

[0011] Preferably, the mass ratio of 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 1.12-1.37:1; more preferably, it is 1.25:1.

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

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

[0015] Preferably, the amino acid accounts for 1.37-3.40% of the mass of the bioconverted bear bile powder.

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

[0017] Preferably, calculated by mass percentage of the bioconverted bear bile powder, the amino acids 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, 0.00-0.00% aspartic acid, and 0.00-0.20% serine.

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

[0019] The biotransformed bear bile powder selected by the present invention is rich in bile acid, and also contains a variety of amino acids and proteins, all of which are edible components, safe ingredients, 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 may be due to the low content of highly acidic components such as 7-keto-TLCA. In addition, the biotransformed bear bile powder of the present invention has good solubility in water, with a solubility of about 17.4g / 100g at room temperature, and has no special smell, and has the necessary conditions for use in cosmetics.

[0020] Preferably, the bioconverted bear bile powder is prepared by the following method:

[0021] S1: Disperse the avian bile powder in glycine buffer, add 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution, adjust the pH, and perform the enzyme conversion reaction for 6 to 18 hours;

[0022] S2: The product after the enzyme conversion reaction in step S1 is subjected to rotary evaporation, washing, concentrating and drying to obtain bioconverted bear bile powder.

[0023] Preferably, the TCDCA content of the poultry gallbladder powder is above 70%.

[0024] Preferably, the fowl gall powder is prepared by the following steps:

[0025] M1: Cut the bird gallbladder to collect bile, place the bile in a 0-4℃ environment to condense for 10-30 hours, filter with a filter membrane with a pore size of 0.25-0.45μm to remove the condensed oil, and retain the liquid phase;

[0026] M2: Concentrate the liquid phase to 1 / 3-1 / 2 of the volume to obtain extract I, add 2-10 times the volume of water, stir evenly at 0-4°C, let stand for 10-30 hours, and filter with a filter membrane with a pore size of 0.25-0.45 μm;

[0027] M3: Concentrate the filtrate to the same volume as extract I to obtain extract II, add 2 to 10 times the volume of 90 to 95% ethanol aqueous solution, stir evenly at 0 to 4°C, let stand for 10 to 30 hours, filter with a filter membrane with a pore size of 0.25 to 0.45 μm, and then concentrate and dry to obtain poultry gall powder.

[0028] Preferably, the stirring speed is 30-80 rpm.

[0029] Preferably, the avian gall powder includes one or more of chicken gall powder, duck gall powder and goose gall powder, which are prepared from chicken gall, duck gall or goose gall respectively by the above method.

[0030] Preferably, the 7α-hydroxysteroid dehydrogenase conversion liquid is prepared by preparing enzyme bacteria A according to the method described in the prior patent CN 111407780A, and each 10 g of enzyme bacteria A is dispersed with 50 mL of glycine buffer.

[0031] Preferably, the 7β-hydroxysteroid dehydrogenase conversion liquid is prepared by preparing enzyme bacteria B according to the method described in the prior patent CN 111407780A, and each 10g of enzyme bacteria B is dispersed with 50mL of glycine buffer.

[0032] Preferably, in the method for preparing the bioconversion bear bile powder, the mass ratio of enzyme bacteria A to enzyme bacteria B is 1:2-10; more preferably, it is 1:6-10.

[0033] Preferably, the volume proportion of the 7α-hydroxysteroid dehydrogenase conversion solution in the reaction solution of the enzyme conversion reaction in step S1 is 3-5%; more preferably, it is 4%;

[0034] Preferably, the volume proportion of the 7β-hydroxysteroid dehydrogenase conversion solution in the reaction solution of the enzyme conversion reaction in step S1 is 30-50%; more preferably, it is 40%.

[0035] The reaction solution of the enzyme conversion reaction refers to a mixture of avian bile powder, glycine buffer, 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution.

[0036] Preferably, the concentration of the glycine buffer is 80-120 mM; more preferably, it is 100 mM.

[0037] Preferably, the concentration of the avian gall powder in the reaction solution of the enzyme conversion reaction in step S1 is 50 to 250 g / L.

[0038] Preferably, the pH is 6 to 9; more preferably, it is 7.5.

[0039] Preferably, the temperature of the enzyme conversion reaction is 20-30°C; more preferably, it is 25°C.

[0040] Preferably, the enzyme conversion reaction time is 14 to 16 hours.

[0041] Preferably, the cosmetic is an antioxidant cosmetic;

[0042] Preferably, the cosmetic has the effect of scavenging free radicals.

[0043] Preferably, the cosmetic is an oil-controlling and acne-removing cosmetic;

[0044] Preferably, the cosmetic is capable of inhibiting sebum secretion;

[0045] Preferably, the cosmetic has an inhibitory effect on sebaceous gland cells.

[0046] Preferably, the cosmetic is a soothing cosmetic;

[0047] Preferably, the cosmetic has an inhibitory effect on the secretion of inflammatory mediators by macrophages.

[0048] Preferably, the cosmetic has an inhibitory effect on mast cell degranulation.

[0049] Preferably, the cosmetics are repair cosmetics;

[0050] Preferably, the cosmetic has a repairing effect on damaged cells.

[0051] In order to further expand the application field of biotransformation bear bile powder and provide new active raw materials for the cosmetics field, the inventors found in the study that the biotransformation bear bile powder prepared by a specific method has multi-faceted skin care activity and exhibits effective antioxidant, oil control, soothing and repairing effects: the biotransformation bear bile powder can scavenge free radicals and exhibit antioxidant activity, thereby further playing an anti-aging and anti-photoaging role; the biotransformation bear bile powder also inhibits the activity of sebaceous gland cells SZ95, reduces the secretion of sebum, and avoids problems such as acne caused by excessive sebum. In particular, the biotransformation bear bile powder only inhibits the activity of SZ95, and hardly produces cytotoxicity to it, and does not affect the normal function of the skin; at the same time, after the application of the biotransformation bear bile powder, the secretion of the inflammatory mediator TNF-α of macrophages is significantly reduced, and the degranulation rate of mast cells is also significantly reduced, thereby exhibiting excellent anti-inflammatory and soothing activity; in addition, for damaged tissue cells, the biotransformation bear bile powder can significantly increase the number of living cells, reduce the loosening of the stratum corneum, and exhibit obvious repair effects on skin that loses tissue vitality after stimulation.

[0052] On the other hand, the present invention provides a cosmetic for the above application, wherein the cosmetic is a lotion, emulsion, facial cream, eye cream, essence, facial mask, facial cleanser, shampoo, shower gel, soap, sunscreen or hand cream; in addition to the bioconverted bear bile powder, the cosmetic also contains auxiliary materials acceptable in the cosmetic field.

[0053] Preferably, the cosmetic is a facial cream, and the components of the facial cream include: stearate, stearyl alcohol, glycerin, water, a thickener, a preservative and biotransformed bear bile powder;

[0054] Preferably, the biotransformed bear bile powder accounts for 2-10% of the mass of the cream;

[0055] Typically, the biotransformed bear bile powder accounts for 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the mass of the cream.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1. The present invention pioneered the use of biotransformed bear bile powder in cosmetics, further expanding the application field of artificial bear bile powder and having great industrial value;

[0058] 2. The biotransformed bear bile powder used in the present invention is low-cost and is selected from edible raw materials. The ingredients are safe and environmentally friendly, cater to the market hotspot of "cosmeceuticals", and meet the preferences of consumers;

[0059] 3. The present invention creatively discovered that the artificial bear bile powder prepared by the biotransformation method has antioxidant activity, can effectively remove free radicals, and has a certain anti-aging and anti-photoaging effect;

[0060] 4. The present invention creatively discovered that biotransformed bear bile powder can inhibit the secretion of sebum by sebaceous gland cells, play an oil control role, and help prevent the occurrence of acne;

[0061] 5. The present invention creatively discovered that biotransformed bear bile powder has anti-inflammatory and soothing effects, and can inhibit the occurrence of inflammation from multiple pathways, thus achieving a soothing effect;

[0062] 6. The present invention creatively discovered that biotransformed bear bile powder has a repairing effect and has obvious effects on the repair and activation of damaged cells in skin tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the relationship between the scavenging rate and concentration of ABTS free radicals by biotransformed bear bile powder;

[0064] Figure 2 This is the relationship between the biotransformation bear bile powder and the cell viability of SZ95 cells and its concentration;

[0065] Figure 3 Fluorescence microscope photo of lipid droplets stained with Nile red in the oil control test of bioconverted bear bile powder;

[0066] Figure 4 This is the relationship between lipid droplet content and concentration in the oil control test of biotransformed bear bile powder;

[0067] Figure 5 This is the relationship between the cell viability and concentration of biotransformed bear bile powder on macrophage RAW264.7;

[0068] Figure 6 This is the relationship diagram between TNF-α content and biotransformation bear bile powder concentration in inflammatory factor detection;

[0069] Figure 7This is the relationship between PGE2 content and biotransformation bear bile powder concentration in inflammatory factor detection;

[0070] Figure 8 This is the relationship between the cell viability and concentration of the biotransformed bear bile powder on mouse mast cell tumor cells P815;

[0071] Fig. 9 These are microscope photos of the degranulation of cells in each group in the mast cell degranulation test;

[0072] Fig.10 This is the relationship between the cell degranulation rate and the concentration of biotransformed bear bile powder in the mast cell degranulation test;

[0073] Fig.11 This is a microscope photo of the skin model after drug administration in the tissue morphology test;

[0074] Fig.12 This is a trend chart of relative tissue vitality in tissue vitality detection. DETAILED DESCRIPTION

[0075] Example 1

[0076] This embodiment provides an application of biotransformed bear bile powder in the preparation of cosmetics. The main components and contents of the biotransformed bear bile powder are shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] The bioconversion bear bile powder of this embodiment is prepared by the following method:

[0081] S1: Disperse 250 g chicken gall powder in 300 mL of 100 mM glycine buffer, add 40 mL of 7α-hydroxysteroid dehydrogenase conversion solution and 400 mL of 7β-hydroxysteroid dehydrogenase conversion solution, add 100 mM glycine buffer to 1 L, adjust the pH to 7.5 with 5 M NaOH aqueous solution, and perform enzyme conversion reaction at 25 °C for 16 h;

[0082] S2: The product after the enzyme conversion reaction in step S1 is rotary evaporated at 65°C to half of the remaining volume, and 10 times the volume of 95% ethanol aqueous solution is added for washing, and the precipitate is removed by centrifugation; the supernatant is evaporated at 75°C to the volume before washing, and vacuum dried at 85°C to obtain bioconverted bear bile powder.

[0083] Wherein, chicken gall powder is prepared by the following method:

[0084] M1: Cut the chicken gallbladder to collect bile, place the bile at 2°C for 24 hours to condense, filter with a filter membrane with a pore size of 0.45 μm to remove the condensed oil, and retain the liquid phase;

[0085] M2: The liquid phase was concentrated to 1 / 2 of the volume by using an external circulation evaporator at 65°C under reduced pressure to obtain extract I, and 10 times the volume of water was added. After stirring evenly at 60 rpm at 2°C, it was allowed to stand for 24 hours and filtered using a filter membrane with a pore size of 0.45 μm;

[0086] M3: The filtrate was concentrated under reduced pressure at 65°C using an external circulation evaporator to the same volume as extract I to obtain extract II, 10 times the volume of 95% ethanol aqueous solution was added, the mixture was stirred evenly at 2°C, and allowed to stand for 24 hours, filtered through a filter membrane with a pore size of 0.45 μm, and then concentrated under reduced pressure at 65°C using an external circulation evaporator and dried to dryness using a vacuum belt dryer at 80°C to obtain chicken gall powder.

[0087] The 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution used in this example are preferred sources in the specification.

[0088] The cosmetics are antioxidant cosmetics.

[0089] The present embodiment also provides a cosmetic for the above application, wherein the cosmetic is a facial cream, and the components of the facial cream, calculated by mass percentage of the facial cream, include 10% white oil, 1% PEG-60, 2.5% propyl heptyl octanoate, 4% cetearyl alcohol, 3% glycerol, 1% xanthan gum, 1.5% undecane (and) tridecane, 0.05% EDTA, 0.5% phenoxyethanol, 3% biotransformed bear bile powder, and the balance is made up with deionized water.

[0090] Example 2

[0091] This embodiment provides an application of biotransformed bear bile powder in the preparation of cosmetics and the cosmetics used in the application. The implementation method is the same as that of Example 1, except that the cosmetics are oil-controlling and anti-acne cosmetics.

[0092] Example 3

[0093] This embodiment provides an application of biotransformed bear bile powder in the preparation of cosmetics and the cosmetics used in the application. The implementation method is the same as that of Example 1, except that the cosmetics are soothing cosmetics.

[0094] Example 4

[0095] This embodiment provides an application of biotransformed bear bile powder in the preparation of cosmetics and the cosmetics used in the application. The implementation method is the same as that of Example 1, except that the cosmetics are repair cosmetics.

[0096] Performance Testing

[0097] 1. Antioxidant test

[0098] ABTS free radical scavenging ability test: 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% using PBS buffer at pH 7.4.

[0099] The C1, C2, T1, and T2 reaction groups were prepared in sequence according to the following system ratios. After incubation at 25°C for 5 min, the absorbance OD index at 734 nm was measured to calculate the ABTS free radical scavenging rate.

[0100]

[0101] Among them, the ABTS working solution comes from the ABTS method of the Biyuntian total antioxidant capacity test kit; the test solution is the biotransformation bear bile powder sample solution of various concentrations prepared above, and the sample solvent is PBS buffer solution with a pH of 7.4; each test solution is set with 3 replicate wells and 1 background control well.

[0102] The calculation method of ABTS free radical scavenging rate is:

[0103]

[0104] Where, C1——blank absorbance value of ABTS system

[0105] C2——Blank absorbance value without ABTS system

[0106] T1——The sample group has the absorbance value of ABTS system

[0107] T2——The sample group has no ABTS system absorbance value.

[0108] The results of ABTS free radical scavenging rate are shown in Table 2 and Figure 1 As shown, BC is the blank control.

[0109] Table 2

[0110]

[0111] Figure 1 In the data, t-test method was used for statistical analysis. The significance of the sample group compared with the BC group was indicated by *, p-value < 0.05 was indicated by *, and p-value < 0.01 was indicated by **.

[0112] 2. Sebaceous gland cell oil control test

[0113] SZ95 cell inhibition test:

[0114] Reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (Sigma), MTT (Sigma), DMSO (Sigma), trypsin (Gibco).

[0115] Sample: Bioconverted bear bile powder of Example 2.

[0116] 2.1 Cytotoxicity test

[0117] 1) Cell seeding: 1×10 4 SZ95 cells were seeded into 96-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2);

[0118] 2) Experimental grouping: The experiment set up a zero adjustment group, a control group, a positive control group and a sample group. In the sample group, each sample was set to 8 gradient concentrations, and 3 replicate wells were set at each concentration. The 8 concentrations are as follows, and the solvent is PBS buffer at pH 7.4;

[0119]

[0120] 3) Administration: Administration was performed when the cell plating rate 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; the zero adjustment group had no cell inoculation and only 200 μL of cell culture medium was added. After administration, the 96-well plate was placed in an incubator (37°C, 5% CO2) for incubation.

[0121] 4) Detection: After the cells were incubated for 24 hours, the supernatant was discarded, and a medium containing 0.5 mg / mL MTT was added. The cells were incubated at 37°C in the dark for 2 hours. After the incubation, the supernatant was discarded, 100 μL DMSO was added to each well, and the OD value was read at 490 nm. The relative cell viability was calculated according to the following formula:

[0122]

[0123] The test results are shown in Table 3 and Figure 2 as shown.

[0124] Table 3

[0125]

[0126] 2.2 Oil control test

[0127] 1) Cell seeding: 1×10 5 SZ95 cells were seeded into 24-well plates at a seeding density of 10 cells / well and incubated overnight in an incubator (37°C, 5% CO2);

[0128] 2) Prepare the working solution for each experimental group as follows:

[0129]

[0130] 3) Drug administration: According to the above experimental design, when the cell plating rate in the 24-well plate reaches 40% to 60%, group drug administration is carried out, and the dosage per well is 1.0 mL. Each group has 3 replicate wells, and the culture is continued in the incubator (37°C, 5% CO2) for 24 hours;

[0131] 4) Discard the culture medium, rinse the cells with PBS, fix them, add Nile red to stain for 15 minutes, rinse them and take pictures with a fluorescence microscope. The pictures are as follows: Figure 3 As shown;

[0132] 5) Result analysis: The fluorescence intensity was quantitatively analyzed using Image Pro Plus software. The lipid droplet content is shown in Table 4 and Figure 4 shown.

[0133] Table 4

[0134]

[0135] Statistical analysis was performed using the t-test method. When the NC group was compared with the BC group, significance was indicated by #, p-value < 0.05 was indicated by #, and p-value < 0.01 was indicated by ##; when the sample group was compared with the NC group, significance was indicated by *, p-value < 0.05 was indicated by *, and p-value < 0.01 was indicated by **.

[0136] 3. Relief test

[0137] 3.1 Macrophage-inflammatory factor detection

[0138] Test cells: RAW264.7 macrophages.

[0139] Main reagents: high-glucose DMEM culture medium (VivaCell), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), LPS (Sigma), and dexamethasone (Sigma).

[0140] 3.1.1 Cytotoxicity test

[0141] Referring to the method in 2.1, the cells were replaced with macrophage RAW264.7 and the MTT toxicity test was performed. The test results are shown in Table 5 and Figure 5 shown.

[0142] Table 5

[0143]

[0144] 3.1.2 Detection of inflammatory factors

[0145] 1) Press 1×10 5 RAW264.7 cells were seeded into 24-well plates at a seeding density of 10 cells / well and incubated overnight in an incubator (37°C, 5% CO2);

[0146] 2) Prepare the working solution for each experimental group as follows:

[0147]

[0148] 3) Drug administration: According to the above experimental design, when the cell plating rate in the 24-well plate reaches 40% to 60%, group drug administration is carried out, and the dosage per well is 1.0 mL. Each group has 3 replicate wells, and the culture is continued in the incubator (37°C, 5% CO2) for 24 hours;

[0149] 4) LPS stimulation: according to the experimental design, add 100 μL of 10×LPS working solution to the wells where the drugs have been administered, shake the wells left and right to mix the drugs in the wells, and the final concentration of LPS is 1 μg / mL. Continue to culture in the incubator (37°C, 5% CO2) for 24 hours;

[0150] 5) Sample collection: After the incubation is completed, the cell culture supernatant is collected in an EP tube and stored in a -80°C refrigerator;

[0151] 6) Detection of TNF-α content: Detection was performed according to the operating instructions of the Mouse TNF-α ELISA kit;

[0152] PGE2 content detection: Detection was performed according to the operating instructions of the Prostaglandin E2 ELISA kit.

[0153] The results of TNF-α content detection are shown in Table 6 and Figure 6 As shown in Table 7 and Figure 7 shown.

[0154] Table 6

[0155]

[0156] Table 7

[0157]

[0158] The definitions of # and * are the same as above.

[0159] 3.2 Mast cell degranulation detection

[0160] Test cells: Mouse mastocytoma cells P815, purchased from the Cell Bank of the Chinese Academy of Sciences.

[0161] Main reagents: high-glucose DMEM culture medium (VivaCell), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), LPS (Sigma), and CCK-8 (Biyuntian).

[0162] 3.2.1 Cytotoxicity test

[0163] Referring to the method in 2.1, the cells were replaced with mouse mastocytoma cells P815, and the MTT toxicity test was performed with a detection wavelength of 450nm. The test results are shown in Table 8 and Figure 8 shown.

[0164] Table 8

[0165]

[0166] 3.2.2 Degranulation rate test method

[0167] 1) Press 1×10 5 P815 cells were seeded into 24-well plates at a seeding density of 10 cells / well and incubated overnight in an incubator (37°C, 5% CO2);

[0168] 2) Prepare the working solution for each experimental group as follows:

[0169]

[0170] 3) Drug administration: According to the above experimental design, when the cell plating rate in the 24-well plate reaches 40% to 50%, group drug administration is carried out, and the dosage per well is 1.0 mL. Each group has 3 replicate wells, and the culture is continued in the incubator (37°C, 5% CO2) for 2 hours;

[0171] 4) C48 / 80 stimulation: After the samples were incubated for 2 hours, the solution was discarded; according to the experimental design, 1 mL of serum-free high-glucose DMEM culture medium was added to the blank control group, 1 mL of serum-free high-glucose DMEM culture medium containing C48 / 80 was added to the negative control group, 1 mL of serum-free high-glucose DMEM culture medium containing C48 / 80 and sodium cromoglycate was added to the positive control group, and 1 mL of serum-free high-glucose DMEM culture medium containing C48 / 80 and the corresponding sample administration concentration was added to the sample group. After the administration, C48 / 80 was stimulated for 45 minutes, and the reaction was terminated in an ice bath;

[0172] 5) Cell morphology observation: After the culture, observe the degranulation of cells in each group under an inverted microscope and take pictures. Fig. 9The photos were counted and the cell degranulation rate was calculated using IPP software. The calculation formula is:

[0173]

[0174] The calculation results are shown in Table 9 and Fig.10 shown.

[0175] Table 9

[0176]

[0177] The definitions of # and * are the same as above.

[0178] 4. Repair test

[0179] Test model: epidermal skin model

[0180] Main reagents:

[0181] Maintenance medium PBS (Gibco), isopropanol (Sinopharm), 4% paraformaldehyde (Sigma).

[0182] The experimental design is as follows:

[0183]

[0184] Preparation of working stock solution:

[0185] 1) Preparation of 0.4% SLS stock solution: weigh 0.0080 g SLS and dissolve it in 2 mL PBS solution, filter through 0.22 μm to prepare 0.4% SLS stock solution for later use;

[0186] 2) Preparation of 0.2% SLS working solution: aspirate 0.5 mL of 0.4% SLS solution and add 0.5 mL of PBS to prepare 0.2% SLS working solution;

[0187] 3) Preparation of positive control 1 (dexamethasone) working solution: Dissolve 100 mg of dry dexamethasone in 1 mL of DMSO to prepare a 100 mg / mL stock solution; pipette 6 μL of the 100 mg / mL stock solution into 6 mL of model culture medium to prepare a 0.01% dexamethasone working solution for later use.

[0188] 4) Preparation of positive control 2 (WY14643) working solution: Weigh 10 mg of WY14643 powder and dissolve it in 1 mL of DMSO to prepare a 30 mM WY14643 stock solution; add 10 μL of WY14643 stock solution (30 mM) to 6 mL of model culture medium to prepare a 50 μM working solution for use.

[0189] Model drug administration:

[0190] 1) According to the above experimental design, transfer the model to a 24-well plate (add 0.9 mL of the model culture medium of the corresponding group in advance) and mark the test group number on the 24-well plate;

[0191] 2) Take 50 μL of the prepared working solution of different groups and add it to the model surface. Add 0.2% SLS solution to the model surface of NC and PC groups, and add working solution of corresponding concentration to the sample group. Use a tweezer to clamp the nylon membrane and cover the surface of each model so that the sample is evenly distributed on the model surface. Place it in an incubator (37°C, 5% CO2) and incubate it for 24 hours.

[0192] 3) After the incubation, use a washing bottle containing sterile PBS solution to clean the test substance remaining on the surface of the model, and use a sterile cotton swab to gently wipe off the residual liquid inside and outside the model.

[0193] 4.1 Tissue morphology test

[0194] The model after drug administration was fixed with 4% paraformaldehyde. After fixation for 24 hours, the model was removed by circumcision and H&E staining was performed. The model was observed under a microscope and pictures were collected. Fig.11 shown.

[0195] Microscopic photos of tissue morphology showed that compared with the BC group, the stratum corneum of the epidermal model in the NC group was loose and thickened, the living cell layer was damaged, the number of living cell layers was reduced, and vacuoles appeared, indicating that the SLS stimulation conditions were effective.

[0196] Compared with the NC group, the four-layer structure boundaries of the PC (WY14643) group model were clear, the living cell layer cells were compactly arranged, the looseness of the stratum corneum was significantly improved, and the number of living cells increased, indicating that this positive control test was effective.

[0197] Compared with the NC group, the loosening of the stratum corneum was significantly improved in the sample biotransformed bear bile powder at a concentration of 3%, and the number of living cells increased significantly.

[0198] 4.2 Tissue Viability Detection

[0199] 1) MTT incubation: After the above-mentioned model was cleaned after administration, a 24-well plate was prepared according to the number of models and marked accordingly, and 1 mL of MTT working solution (1 mg / mL) was added to each well; the 24-well plate containing the model was placed in an incubator (37° C., 5% CO2) and incubated for 1 h;

[0200] 2) Isopropanol extraction: After the MTT incubation, wash the outer surface of the model with PBS, wipe it dry with a sterile cotton swab, transfer it to a new 24-well plate, and mark it; add 2 mL of isopropanol to the inner surface of the model, seal the 24-well plate with a sealing film, and shake it on a plate shaker for 2 hours;

[0201] 3) Reading: Pierce the model with a pipette tip to allow the isopropanol extract to flow out of the model into the 24-well plate; discard the pierced model, blow the isopropanol extract in each well 3 times to mix it thoroughly; after mixing, draw 200 μL of isopropanol extract from each well, add it to the corresponding wells of the 96-well plate, and mark it; read the absorbance (OD) value at a wavelength of 570 nm using an ELISA reader;

[0202] 4) Calculation: The relative tissue activity of each experimental group was calculated according to the following formula. The results are recorded in Table 10 and the trend graph is shown in Fig.12 shown.

[0203]

[0204] Table 10

[0205]

[0206] The definitions of # and * are the same as above.

[0207] 5. Toxicology Experiment

[0208] According to the test and evaluation methods in Chapter 6 of the "Technical Specification for Safety of Cosmetics" (2015 edition), the biotransformed bear bile powder in Example 1 of the present invention was subjected to an acute oral toxicity test, an acute dermal toxicity test, an acute eye irritation test, a multiple skin irritation test, a skin allergy test, a skin phototoxicity test, a skin photoallergy test, and an in vitro mammalian cell chromosome aberration test. The toxicology test results are as follows:

[0209] 5.1 Acute oral toxicity test: The oral LD50 of biotransformed bear bile powder to SD rats is > 5000 mg / kg body weight, which is practically non-toxic. The weight and mortality of the poisoned animals (10 SPF-grade SD rats) are shown in Table 11.

[0210] Table 11

[0211]

[0212] 5.2 Acute dermal toxicity test: The dermal LD50 of biotransformation bear bile powder to SD rats is ≥2180 mg / kg body weight, which is slightly toxic. The body weight and mortality of the poisoned animals (10 SPF-grade SD rats) are shown in Table 12.

[0213] Table 12

[0214]

[0215] 5.3 (No flushing) Acute eye irritation test: The biotransformed bear bile powder showed mild irritation, and the eye irritation scores of three New Zealand rabbits are shown in Table 13. In this test, the single application amount of the biotransformed bear bile powder to a single individual was 0.1 g.

[0216] Table 13

[0217]

[0218] Note: The mean integral value is rounded to 2 decimal places.

[0219] 5.4 Multiple skin irritation test: The biotransformed bear bile powder showed no irritation, and the skin irritation scores of 4 New Zealand white rabbits are shown in Table 14. In this test, the single application amount of the biotransformed bear bile powder to a single individual was 0.5 g.

[0220] Table 14

[0221]

[0222] 5.5 Skin allergy test (BT method): The skin allergy test of biotransformation bear bile powder on albino guinea pigs showed no skin allergy. The skin reaction intensity of albino guinea pigs is shown in Table 15, wherein the negative control was distilled water applied during induction contact and 0.2g of biotransformation bear bile powder was applied during stimulation contact; the positive control was 0.8% 2,4-dinitrochloropropiophenone acetone solution applied during induction contact and 0.2% 2,4-dinitrochloropropiophenone acetone solution applied during stimulation contact; the test substance group was 0.2g of biotransformation bear bile powder applied during induction contact and 0.2g of biotransformation bear bile powder applied during stimulation contact.

[0223] Table 15

[0224]

[0225] 5.6 Skin phototoxicity test: The skin phototoxicity test of biotransformed bear bile powder on adult albino guinea pigs showed no skin phototoxicity, and the skin reaction scores of guinea pigs are shown in Table 16. In this test, the application rate of biotransformed bear bile powder to a single individual and a single area was 0.2g.

[0226] Table 16

[0227]

[0228] The 1, 2, 3, and 4 in the skin reaction score table represent different experimental treatments of the four hair removal areas of the same animal; 1 means application of biotransformation bear bile powder but no irradiation; 2 means application of biotransformation bear bile powder and irradiation; 3 means no application of biotransformation bear bile powder and no irradiation; 4 means no application of biotransformation bear bile powder and irradiation.

[0229] 5.7 Skin photoallergy test: The skin photoallergy test of biotransformation bear bile powder on albino guinea pigs showed no skin photoallergy, and the skin reaction scores of albino guinea pigs during the light stimulation stage are shown in Table 17. In this test, 0.1g of biotransformation bear bile powder was moistened with saline and then applied to a single individual and a single area during the light induction stage; 0.02g of biotransformation bear bile powder was moistened with saline and then applied to a single individual and a single area during the light stimulation stage.

[0230] Table 17

[0231]

[0232] In the skin reaction score of the light stimulation stage, 1, 2, 3, and 4 are Appendix 2 of the Notice No. 136 of the State Food and Drug Administration in 2017 of the "Technical Specifications for Safety of Cosmetics" (2015 Edition). Figure 2 During the photoexcitation phase, the test substance is applied to four areas exposed to ultraviolet light.

[0233] 5.8 In vitro mammalian cell chromosome aberration test: Biotransformation bear bile powder is non-mutagenic, and the chromosome aberration rate is shown in Table 18. In this test, the cell line, metabolite activation system (liver microsomal enzyme mixture-S9 mixture) and positive substances are all derived from in vitro chromosome aberration reagent bottles, and the supplier is Beijing Huizhi Taikang, and the reagent bottle batch number is 0221011 / 0221012.

[0234] Table 18

[0235]

[0236] Note: Use χ 2 Statistical analysis was performed by test, and compared with the negative control group, *P<0.05.

Claims

1. An application of biotransformed bear bile powder in the preparation of cosmetics, characterized in that: The biotransformed bear bile powder contains bile acid; the bile acid includes tauroursodeoxycholic acid, taurochenodeoxycholic acid and cholic acid.

2. The use of the biotransformed bear bile powder in the preparation of cosmetics according to claim 1, characterized in that: The mass fraction of bile acid in the bioconversion bear bile powder is 60-75%.

3. The use of the biotransformed bear bile powder according to claim 1 or 2 in the preparation of cosmetics, characterized in that: The mass ratio of tauroursodeoxycholic acid, taurochenodeoxycholic acid and cholic acid is 1.1-1.5:1:0.3-0.

5.

4. The use of the biotransformed bear bile powder according to claim 1 or 2 in the preparation of cosmetics, characterized in that: The biotransformed bear bile powder also contains amino acids, proteins and trace elements.

5. The use of the biotransformed bear bile powder in the preparation of cosmetics according to claim 1, characterized in that: The biotransformed bear bile powder is prepared by the following method: S1: Disperse the avian bile powder in glycine buffer, add 7α-hydroxysteroid dehydrogenase conversion solution and 7β-hydroxysteroid dehydrogenase conversion solution, adjust the pH, and perform the enzyme conversion reaction for 6 to 18 hours; S2: The product after the enzyme conversion reaction in step S1 is subjected to rotary evaporation, washing, concentrating and drying to obtain bioconverted bear bile powder.

6. The use of the biotransformed bear bile powder according to claim 1 or 5 in the preparation of cosmetics, characterized in that: The cosmetics are antioxidant cosmetics.

7. The use of the biotransformed bear bile powder according to claim 1 or 5 in the preparation of cosmetics, characterized in that: The cosmetics are oil-controlling and acne-removing cosmetics.

8. The use of the biotransformed bear bile powder according to claim 1 or 5 in the preparation of cosmetics, characterized in that: The cosmetics are soothing cosmetics.

9. The use of the biotransformed bear bile powder according to claim 1 or 5 in the preparation of cosmetics, characterized in that: The cosmetics are repairing cosmetics.

10. A cosmetic for use according to any one of claims 1 to 9, characterized in that: The cosmetics are lotions, emulsions, face creams, eye creams, essences, facial masks, facial cleansers, shampoos, shower gels, soaps, sunscreens or hand creams; in addition to biotransformed bear bile powder, the cosmetics also contain auxiliary materials acceptable in the cosmetics field.

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