Application of sparassis crispa polysaccharide in lip care product

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to application of sparassis crispa polysaccharide in lip care products. The applicant creatively discovers that the sparassis crispa polysaccharide has the effect of promoting lip epithelial cell proliferation, and the effect of the sparassis crispa polysaccharide is far better than that of the sparassis crispa polysaccharide for promoting skin keratinocyte proliferation, so that the sparassis crispa polysaccharide is more suitable for lip protection compared with skin protection. In addition, the sparassis crispa polysaccharide also has the effects of moisturizing, moistening, repairing, resisting wrinkles and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and in particular relates to application of hydrangea polysaccharide in lip care products. Background Art

[0002] Human lips, including upper and lower lips, are red lips, which are the places where women apply lipstick or lipstick in daily life. Red lips are the transition area between facial skin and oral mucosa. The skin is extremely thin and has no glands or pigments, so the red color of the lips mainly comes from the abundant blood vessels under the skin, making them look red and soft.

[0003] The main lip problems are dryness and aging, of which dryness is taken seriously, while aging is neglected. The thickness of the lip skin is only 1 / 3 of the skin on other parts of the body, which makes the lips appear reddish. When the lips are drier, their appearance color will be lighter. Since the red lips lack sweat glands and sebaceous glands and have a weak moisturizing barrier, their moisture is maintained entirely by the abundant local capillaries and a small number of underdeveloped sebaceous glands, making them very easy to dry out. The lips lack melanocytes and melanin themselves, and lack self-protection against ultraviolet rays. The lower lip, in particular, is more susceptible to exposure to sunlight, resulting in ultraviolet damage / photoaging, reduced lip tissue volume (shrinkage) and wrinkles, which means that the lips are prone to aging.

[0004] At present, lip care mainly uses lip cosmetics to relieve lip dryness through exogenous hydration or film-forming moisturizing. There is still a lack of measures for endogenous moisturizing of the lips or delaying lip aging. In theory, lip cosmetics can not only adjust the skin color and appearance of the lips, such as lipstick and lip gloss, but also protect the lips to keep them soft and shiny, prevent dryness and aging, such as lipstick and lip mask. At present, lip care mainly relies on exogenous methods to moisturize the lips, rarely uses endogenous methods to moisturize, and rarely considers the problem of lip aging.

[0005] Among them, there are two main ways of exogenous moisturizing of lips:

[0006] (1) Hydration and moisturizing: By using skin care products with high water content from the outside, the moisture content of the skin can be directly increased to achieve the effect of moisturizing the skin.

[0007] (2) Film-forming moisturizing: A protective film composed mainly of wax and oil is formed on the skin surface through various means to prevent the evaporation of skin moisture and the loss of sebum, thereby increasing the skin's moisture content.

[0008] Therefore, there is a need in the art for a lip care product that can protect the lips in an endogenous manner, such as anti-aging and moisturizing the lips. Summary of the invention

[0009] In view of this, in a first aspect, the present invention provides the use of Spadeacanga polysaccharide in lip care products.

[0010] The applicant creatively discovered that hydrangea polysaccharide has the effect of promoting the proliferation of lip epithelial cells, which is much better than the effect of hydrangea polysaccharide in promoting the proliferation of skin keratinocytes. Therefore, it is more suitable for lip care than skin care. In addition, hydrangea polysaccharide also has the effects of moisturizing, nourishing, repairing, and anti-wrinkle.

[0011] In the present invention, the term "lip care product" refers to a product for maintaining the health and beauty of lips, which can have a repairing effect on the lips.

[0012] Furthermore, the lip care is achieved by promoting the proliferation of lip epithelial cells.

[0013] Furthermore, the mass fraction of the hydrangea polysaccharide in the lip care product is 0.001% to 5%, preferably 0.01 to 1%, and more preferably 0.1%.

[0014] Furthermore, the hydrangea polysaccharide is obtained by alcohol extraction, water extraction, carbon dioxide supercritical extraction, enzyme extraction, microwave extraction, ultrasound-assisted extraction, resin method, etc. of hydrangea fruiting bodies. Preferably, the hydrangea polysaccharide is obtained by ethanol extraction of hydrangea fruiting bodies.

[0015] Furthermore, the hydrangea polysaccharide is obtained by crushing hydrangea fruiting bodies, adding liquid to dissolve, hot extraction, filtering, and ethanol precipitation.

[0016] Furthermore, the material-liquid ratio of the added liquid for dissolution is 1:20 (g / mL) to 1:80 (g / mL), preferably 1:30 (g / mL) to 1:70 (g / mL), more preferably 1:40 (g / mL) to 1:60 (g / mL), and most preferably 1:50 (g / mL). That is, 1g of crushed hydrangea fruiting body is added with 50mL of liquid, and the liquid is preferably purified water.

[0017] Further, the temperature of the thermal extraction is 90 to 110° C., preferably 100° C. Further, the extraction time of the thermal extraction is 1 to 3 hours, preferably 2 hours.

[0018] Using various parameters of the above extraction method can make the extraction of hydrangea polysaccharide higher in purity and yield.

[0019] Furthermore, the lip care product can be a lipstick, lip gloss, lip glaze, lip balm, or lip mask.

[0020] In a second aspect, the present invention provides a lip care product, which includes hydrangea polysaccharide, and oils and waxes, wherein the mass fraction of the oils and waxes in the entire lip care product is greater than 30%.

[0021] Furthermore, the mass fraction of the oil and wax substances in the entire lip care product is greater than 50%.

[0022] Furthermore, the oil and wax substances account for 30% to 99% of the mass fraction of the entire lip care product.

[0023] Furthermore, the oil and wax substances account for 50% to 99% of the mass fraction of the entire lip care product.

[0024] The mass fraction of the hydrangea polysaccharide in the lip care product is 0.01% to 5%, preferably 0.1 to 1%, and more preferably 0.1%.

[0025] Furthermore, the lip care product can be a lipstick, lip gloss, lip cream, lip balm, or lip mask.

[0026] Furthermore, the lip care product also includes at least one of a flavor, an antioxidant, an emollient, a filler, a colorant, a preservative, a binder, or a thickener.

[0027] In some specific embodiments, the present invention provides a lip care product, which is a lipstick, including isooctyl palmitate, phenyl isopropyl polydimethicone, plant lanolin, octyldodecanol, polyisobutylene, PEG-10 polydimethicone, olive oil, silica, bentonite, deionized water, hydrangea polysaccharide, 1,3-butylene glycol, disodium EDTA, color paste, antioxidant, preservative, and flavor.

[0028] In some specific embodiments, the present invention provides a lip care product, which is a lip gloss, including glyceryl diisostearate, macadamia oil, No. 70 white oil, propyl dimethicone, vitamin E, glyceryl isooctanoate, polydimethylsiloxane, refined ozokerite, lanolin hydroxystearate, microcrystalline wax, essence, red iron oxide, purified water, and hydrangea polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The negative control was 0.9% NaCl, and the positive controls were 0.2%, 0.3%, and 0.5% sodium hydroxide solutions for CAM results;

[0030] Figure 2 This is the CAM result of the hydrangea polysaccharide sample of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] The term "Sparassis crispa polysaccharide" refers to a polysaccharide derived from the fruiting body of Sparassis crispa Fr. (Amacrosporales, Hydrangeales, Sparassisaceae, Sparassis genus).

[0033] Example 1. Extraction of Hydrangea polysaccharide

[0034] First, the dry fruiting body of Hydrangea serrata was crushed into 10 meshes, and distilled water was added at a solid-liquid ratio of 1:50 g / mL. The mixture was hot-extracted at 110°C for 3 hours, filtered through 300 meshes, and the filtrate was concentrated to 1 / 5 of the original volume at 50°C under reduced pressure. The concentrated polysaccharide solution was precipitated overnight with 4 times the volume of anhydrous ethanol (final concentration 50%, v / v). The precipitate was collected by centrifugation at 5000 r / min for 10 minutes, washed with anhydrous ethanol and acetone, and freeze-dried to obtain the Hydrangea serrata polysaccharide extract.

[0035] Example 2: Lip protection effect of Hydrangea polysaccharide

[0036] Experimental Materials

[0037] Cells: Primary human oral epithelial cells and HaCaT immortalized skin keratinocytes, Zhejiang Meisen Cell Technology Co., Ltd.

[0038] Experimental instruments: electronic balance ME204E, Mettler; carbon dioxide incubator HF90, Healforce; biological safety cabinet 1500LC, Healforce; inverted microscope CKX53, OLYMPUS; centrifuge L3-5K, Eppendorf; cell counter Countess3, Thermo Fisher; multifunctional microplate reader EPLEX, Tecan; refrigerator YCD-EL300, Meiling; constant temperature water bath HWS-24, Shanghai Yiheng; adjustable pipette 10 / 100 / 200 / 1000 / 5000ul, Eppendorf.

[0039] Experimental reagents and consumables: DMEM high-glucose medium, Gibco; fetal bovine serum (FBS), Gibco; trypsin, Biosharp / Gibco; PBS phosphate buffer, Solebao; penicillin-streptomycin mixture (double antibody), ZetaLife; culture flask, BIOFIL; 96-well plate, Afiy; 1.5mL centrifuge tube, Afiy; dimethyl sulfoxide cell culture grade (DMSO), Solebao; glycerol, bulk; CCK8, ZetaLife; AQP3 Elisa Kits, Shanghai ELISA.

[0040] Experimental reagent preparation

[0041] 1) Complete culture medium: Add FBS to DMEM high-glucose culture medium to make the content 10%, double antibody 1%, and store at 4°C. The validity period is 30 days.

[0042] 2) Serum-free culture medium: Add 1% of double antibody to DMEM high-glucose culture medium and store at 4°C. The shelf life is 30 days.

[0043] 3) Test samples: should be prepared on the day of the test, preferably within 4 hours before the test.

[0044] -Liquid samples: need to be filtered and sterilized, then diluted with cell culture medium to the corresponding concentration, generally starting from 10% and diluted to 6-12 concentration gradients;

[0045] - Water-soluble solid samples: first dissolve to a high concentration with complete culture medium, filter and sterilize, then dilute to the corresponding concentration with complete culture medium;

[0046] - Water-insoluble samples: Dissolve to a high concentration with DMSO first, then dilute to the corresponding concentration with complete culture medium. Ensure that the DMSO concentration in the final culture medium does not exceed 1%.

[0047] 4) Positive control: 10% FBS.

[0048] Experimental methods

[0049] Cytotoxicity test

[0050] 1) Cell plating: Select cells that have been passaged for more than 2 generations after recovery for the experiment. Discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 5 mL of complete culture medium and resuspend to count the cells. Dilute the cell suspension to a density of 1×10 5Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0051] 2) Sample addition: Take out the 96-well plate, discard the supernatant, add complete culture medium to the normal control group, add complete culture medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 hours;

[0052] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value at 450nm (minus the OD value at 600nm);

[0053] Cell viability calculation formula:

[0054] Cell viability (%) = sample OD value / blank OD value × 100%

[0055] Subsequent efficacy evaluation experiments were conducted on samples with a safe concentration of more than 90% cell viability.

[0056] Efficacy evaluation

[0057] 1) Cell plating: discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off under a microscope, add 5 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 1 mL of complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0058] Note: A blank control group, a test sample group and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0059] 2) Sample addition: After 24 hours of culture, the cell culture medium was replaced with serum-free culture medium containing the test sample, 200 μL / well, and culture was continued for 24 hours. The positive control group was complete culture medium containing 10% FBS.

[0060] 3) Detection: Remove 100 μL / well of the supernatant in the 96-well plate, add 10 μL / well CCK8 reagent to each well, incubate in a 37°C incubator for 1 to 4 hours, and detect the absorbance value at 450 nm with an enzyme marker. Calculate the sample's ability to increase the proliferation of human oral epithelial cells.

[0061] 3.3 Experimental Results and Statistics

[0062] The calculation formula is as follows: Proliferation rate % = (OD value of experimental group - OD value of blank control group) / (OD value of blank control group - OD value of blank well) × 100%

[0063] The experimental data were plotted using Graphpad Prism software, and the data between multiple groups were analyzed using one-way ANOVA, or the comparison between groups was analyzed using t-test statistics. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference.

[0064] Experimental Results

[0065] As shown in Table 1, 1 mg / ml concentration of hydrangea polysaccharide can significantly promote the proliferation of lip epithelial cells, with a proliferation rate of 10.15%; the positive control 10% FBS can significantly promote the proliferation of lip epithelial cells; as shown in Table 2, 1 mg / ml concentration of hydrangea polysaccharide has no significant effect on the proliferation of skin keratinocytes; the positive control 10% FBS can significantly promote the proliferation of skin keratinocytes. This indicates that hydrangea polysaccharide has a stronger effect on promoting the proliferation of lip epithelial cells than skin keratinocytes, and is more suitable for lip care.

[0066] Table 1. Effects of samples on the proliferation of lip epithelial cells

[0067] sample concentration Proliferation rate %-AVG Standard Deviation % P-value DMEM 10% FBS 78.35 9.88 P<0.05 Hydrangea polysaccharide 1mg / ml 10.15 1.59 P<0.05

[0068] Compared with the blank control group, P<0.05;

[0069] Table 2. Effects of samples on skin keratinocyte proliferation

[0070] sample concentration Proliferation rate %-AVG Standard Deviation % P-value DMEM 10% FBS 56.07 2.51 P<0.05 Hydrangea polysaccharide 1mg / ml 2.47 1.41 P>0.05

[0071] Compared with the blank control group, P<0.05;

[0072] Experimental conclusion

[0073] Under the experimental conditions, hydrangea polysaccharide at a concentration of 1 mg / ml has potential endogenous lip protection effect, which is manifested by significantly promoting the proliferation of lip epithelial cells, and its effect is much better than the proliferation effect on skin keratinocytes, with a difference of about 4 times. This shows that hydrangea polysaccharide protects the lips much better than the skin.

[0074] Example 3: Lip moisturizing effect of Hydrangea polysaccharide

[0075] Cells: Human keratinocytes (HaCaT), Zhejiang Meisen Cell Technology Co., Ltd. CTCC-002-0012.

[0076] Experimental instruments: electronic balance ME204E, Mettler; carbon dioxide incubator HF90, Healforce; biological safety cabinet 1500LC, Healforce; inverted microscope CKX53, OLYMPUS; centrifuge L3-5K, Eppendorf; cell counter Countess3, Thermo Fisher; multifunctional microplate reader EPLEX, Tecan; refrigerator YCD-EL300, Meiling; constant temperature water bath HWS-24, Shanghai Yiheng; adjustable pipette 10 / 100 / 200 / 1000 / 5000ul, Eppendorf.

[0077] Experimental reagents and consumables: DMEM high-glucose medium, Gibco; fetal bovine serum (FBS), Gibco; trypsin, Biosharp / Gibco; PBS phosphate buffer, Solebao; penicillin-streptomycin mixture (double antibody), ZetaLife; culture flask, BIOFIL; 96-well plate, Afiy; 1.5mL centrifuge tube, Afiy; dimethyl sulfoxide cell culture grade (DMSO), Solebao; glycerol, bulk; CCK8, ZetaLife; AQP3 Elisa Kits, Shanghai ELISA.

[0078] Experimental reagent preparation

[0079] 1) Complete culture medium: Add FBS to DMEM high-glucose culture medium to make the content 10%, double antibody 1%, and store at 4°C. The validity period is 30 days.

[0080] 2) Serum-free culture medium: Add 1% of double antibody to DMEM high-glucose culture medium and store at 4°C. The shelf life is 30 days.

[0081] 3) Test samples: should be prepared on the day of the test, preferably within 4 hours before the test.

[0082] -Liquid samples: need to be filtered and sterilized, then diluted with cell culture medium to the corresponding concentration, generally starting from 10% and diluted to 6-12 concentration gradients;

[0083] - Water-soluble solid samples: first dissolve to a high concentration with complete culture medium, filter and sterilize, then dilute to the corresponding concentration with complete culture medium;

[0084] - Water-insoluble samples: Dissolve to a high concentration with DMSO first, then dilute to the corresponding concentration with complete culture medium. Ensure that the DMSO concentration in the final culture medium does not exceed 1%.

[0085] 4) Positive control: 3% glycerol.

[0086] Cytotoxicity test

[0087] 1) Cell plating: Select cells that have been passaged for more than 2 generations after recovery for the experiment. Discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 5 mL of complete culture medium and resuspend to count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0088] 2) Sample addition: Take out the 96-well plate, discard the supernatant, add complete culture medium to the normal control group, add complete culture medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 hours;

[0089] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula:

[0090] Cell viability (%) = sample OD value / blank OD value × 100%

[0091] Subsequent efficacy evaluation experiments were conducted on samples with a safe concentration of more than 90% cell viability.

[0092] Efficacy evaluation

[0093] 1) Cell plating: discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off under a microscope, add 5 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 1 mL of complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0094] Note: A blank control group, a test sample group and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0095] 2) Sample addition: After 24 hours of culture, the cell culture medium was replaced with the culture medium containing the test sample, 200 μL / well, and culture was continued for 24 hours. The positive control group was a complete culture medium containing 3% glycerol.

[0096] 3) Detection: Place the 96-well plate at 4°C, centrifuge at 1000g for 10 minutes, take 100μL of the supernatant from each well and place it on a new 96-well plate for immediate detection (or freeze it in a -20°C refrigerator and test it within one week). Prepare the required standards, antibodies, horseradish peroxidase-labeled avidin working solution, colorimetric solution and stop solution, etc.; dilute the sample according to the protein detection range of the kit to make it suitable for the standard curve, centrifuge at 1000g for 10 minutes, add it to the ELISA plate, add HRP 100ul / well at the same time, and incubate it in a 37°C incubator for 60 minutes; discard the supernatant, wash it 5 times with the prepared washing solution; add staining solution A and B for color development for 15min to 25min; finally, add the stop solution, detect the absorbance value at 450nm with an ELISA reader within 15min, and calculate the AQP3 protein expression level.

[0097] Experimental results and statistics

[0098] The calculation formula is as follows: AQP3 upregulation rate % = (AQP3 content in the experimental group - AQP3 content in the blank control group) / (AQP3 content in the blank control group) × 100%

[0099] The experimental data were plotted using Graphpad Prism software, and the data between multiple groups were analyzed using one-way ANOVA, or the comparison between groups was analyzed using t-test statistics. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference.

[0100] Experimental Results

[0101] As shown in Table 3, hydrangea polysaccharide at a concentration of 0.25% can promote the expression of AQP-3 protein in Hacat cells, with an upregulation rate of 54.43% (P<0.001);

[0102] Table 3 Effects of Hydrangea polysaccharide on AQP-3 protein expression in Hacat cells

[0103]

[0104] Compared with the control group, P<0.001

[0105] Experimental conclusion

[0106] Under the experimental conditions, hydrangea polysaccharide at a concentration of 0.25% had potential endogenous moisturizing effect, which was manifested by promoting the expression of AQP-3 protein in Hacat cells.

[0107] Example 4: Moisturizing / repairing effects of Hydrangea polysaccharide

[0108] Aquaporin 3 (AQP3) is a water molecule transport-related protein with a high content in the skin. In addition to water molecules, AQP3 is also involved in the transport of glycerol, which plays a very important role in maintaining the necessary moisture, elasticity, barrier function and wound healing of the skin. AQP3 is widely distributed in cells of all layers of the skin, including dermal fibroblasts, which also express a small amount of AQP3. Chronic UVA radiation activates fibroblast autophagy in the short term, which may be a compensation for the adaptive changes of fibroblasts. Fibroblasts can partially antagonize the light damage caused by UVA radiation by upregulating AQP3 expression, which is achieved by promoting autophagy. Therefore, quantifying the AQP3 protein expression level of fibroblast membrane components by enzyme-linked immunosorbent assay (ELISA) can reflect the deep moisturizing and repair protection effect of cosmetic raw materials / products on skin damage.

[0109] Experimental Materials

[0110] Cells: Human skin fibroblasts (HFF-1), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0111] Experimental instruments: electronic balance ME204E, Mettler; carbon dioxide incubator HF90, Healforce; biological safety cabinet 1500LC, Healforce; inverted microscope CKX53, OLYMPUS; centrifuge L3-5K, Eppendorf; cell counter Countess3, Thermo Fisher; multifunctional microplate reader EPLEX, Tecan; refrigerator YCD-EL300, Meiling; constant temperature water bath HWS-24, Shanghai Yiheng; adjustable pipette 10 / 100 / 200 / 1000 / 5000ul, Eppendorf.

[0112] Experimental reagents and consumables: DMEM high-glucose medium, Gibco; fetal bovine serum (FBS), Gibco; trypsin, Biosharp / Gibco; PBS phosphate buffer, Solebao; penicillin-streptomycin mixture (double antibody), ZetaLife; culture flask, BIOFIL; 96-well plate, Afiy; 1.5mL centrifuge tube, Afiy; dimethyl sulfoxide cell culture grade (DMSO), Solebao; glycerol, bulk; CCK8, ZetaLife; AQP3 Elisa Kits, Shanghai ELISA.

[0113] Experimental reagent preparation

[0114] 1) Complete culture medium: Add FBS to DMEM high-glucose culture medium to make the content 10%, double antibody 1%, and store at 4°C. The validity period is 30 days.

[0115] 2) Serum-free culture medium: Add 1% of double antibody to DMEM high-glucose culture medium and store at 4°C. The shelf life is 30 days.

[0116] 3) Test samples: should be prepared on the day of the test, preferably within 4 hours before the test.

[0117] -Liquid samples: need to be filtered and sterilized, then diluted with cell culture medium to the corresponding concentration, generally starting from 10% and diluted to 6-12 concentration gradients;

[0118] - Water-soluble solid samples: first dissolve to a high concentration with complete culture medium, filter and sterilize, then dilute to the corresponding concentration with complete culture medium;

[0119] - Water-insoluble samples: Dissolve to a high concentration with DMSO first, then dilute to the corresponding concentration with complete culture medium. Ensure that the DMSO concentration in the final culture medium does not exceed 1%.

[0120] 4) Positive control: 3% glycerol.

[0121] Experimental methods

[0122] Cytotoxicity test

[0123] 1) Cell plating: Select cells that have been passaged for more than 2 generations after recovery for the experiment. Discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 5 mL of complete culture medium and resuspend to count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0124] 2) Sample addition: Take out the 96-well plate, discard the supernatant, add complete culture medium to the normal control group, add complete culture medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 hours;

[0125] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula:

[0126] Cell viability (%) = sample OD value / blank OD value × 100%

[0127] Subsequent efficacy evaluation experiments were conducted on samples with a safe concentration of more than 90% cell viability.

[0128] Efficacy evaluation

[0129] 1) Cell plating: discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off under a microscope, add 5 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 1 mL of complete culture medium to resuspend and count the cells. Dilute the cell suspension to a density of 1×10 5 Each well was inoculated into a 96-well plate at 100 μL / well and incubated overnight in a 37°C, 5% CO2 incubator.

[0130] Note: A blank control group, a test sample group and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0131] 2) Sample addition: After 24 hours of culture, the cell culture medium was replaced with the culture medium containing the test sample, 200 μL / well, and culture was continued for 24 hours. The positive control group was a complete culture medium containing 3% glycerol.

[0132] 3) Detection: Place the 96-well plate at 4°C, centrifuge at 1000g for 10 minutes, take 100μL of the supernatant from each well and place it on a new 96-well plate for immediate detection (or freeze it in a -20°C refrigerator and test it within one week). Prepare the required standards, antibodies, horseradish peroxidase-labeled avidin working solution, colorimetric solution and stop solution, etc.; dilute the sample according to the protein detection range of the kit to make it suitable for the standard curve, centrifuge at 1000g for 10 minutes, add it to the ELISA plate, add HRP 100ul / well at the same time, and incubate it in a 37°C incubator for 60 minutes; discard the supernatant, wash it 5 times with the prepared washing solution; add staining solution A and B for color development for 15min to 25min; finally, add the stop solution, detect the absorbance value at 450nm with an ELISA reader within 15min, and calculate the AQP3 protein expression level.

[0133] Experimental results and statistics

[0134] The calculation formula is as follows: AQP3 upregulation rate % = (AQP3 content in the experimental group - AQP3 content in the blank control group) / (AQP3 content in the blank control group) × 100%

[0135] The experimental data were plotted using Graphpad Prism software, and the data between multiple groups were analyzed using one-way ANOVA, or the comparison between groups was analyzed using t-test statistics. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference.

[0136] Experimental Results

[0137] As shown in Table 4, 0.25% concentration of Hydrangea polysaccharide can promote the expression of AQP-3 protein in HHF-1 cells, with an upregulation rate of 9.8% (P<0.05), which is basically equivalent to 3% glycerol.

[0138] Table 4 Effect of Hydrangea polysaccharide on AQP-3 protein expression in HFF-1 cells

[0139] Group concentration AQP-3 content (ng / ml) Increase rate (%) Blank control group / 1812.12±29.96 / Hydrangea polysaccharide 0.25％ 1989.18±73.18* 9.8％ glycerin 3％ 2026.3±40.3* 11.8％

[0140] Compared with the blank control group, P<0.05

[0141] Experimental conclusion

[0142] Under the experimental conditions, hydrangea polysaccharide at a concentration of 0.25% had potential moisturizing and repairing effects, which was manifested by promoting the expression of AQP-3 protein in HFF-1 cells.

[0143] Example 5: Effect of promoting sebum secretion or moisturizing effect of hydrangea polysaccharide

[0144] Cells: Human sebaceous gland cells, Zhejiang Meisen.

[0145] Experimental instruments: electronic balance ME204E, Mettler; carbon dioxide incubator HF90, Healforce; biological safety cabinet 1500LC, Healforce; inverted microscope (with fluorescence) CKX53, OLYMPUS; centrifuge L3-5K, Eppendorf; cell counter Countess3, Thermo Fisher; multifunctional microplate reader EPLEX, Tecan;

[0146] Refrigerator YCD-EL300, Meiling; Constant temperature water bath HWS-24, Shanghai Yiheng; Adjustable pipette 10 / 100 / 200 / 1000 / 5000μL, Eppendorf;

[0147] Experimental reagents and consumables: DMEM high glucose medium, Gibco; fetal bovine serum (FBS), Gibco; trypsin, Biosharp / Gibco; PBS phosphate buffer, Solebao; penicillin-streptomycin mixture (double antibody), ZetaLife; culture flask, BIOFIL; 96-well plate, Afiy; 1.5 mL centrifuge tube, Afiy; dimethyl sulfoxide cell culture grade (DMSO), Solebao; Nile red, Sigma; CCK8, ZetaLife; DHT, APE×BIO;

[0148] Experimental reagent preparation

[0149] 1) Complete culture medium: Add FBS to DMEM high-glucose culture medium to make the content 10%, double antibody 1%, and store at 4°C. The validity period is 30 days.

[0150] 2) Serum-free culture medium: Add 1% of double antibody to DMEM high-glucose culture medium and store at 4°C. The shelf life is 30 days.

[0151] 3) Test samples: should be prepared on the day of the test, preferably within 4 hours before the test.

[0152] -Liquid samples: need to be filtered and sterilized, then diluted with cell culture medium to the corresponding concentration, generally starting from 10% and diluted to 6-12 concentration gradients;

[0153] - Water-soluble solid samples: first dissolve to a high concentration with complete culture medium, filter and sterilize, then dilute to the corresponding concentration with complete culture medium;

[0154] - Water-insoluble samples: Dissolve to a high concentration with DMSO first, then dilute to the corresponding concentration with complete culture medium. Ensure that the DMSO concentration in the final culture medium does not exceed 1%.

[0155] 4) Positive control: 0.5 mg / mL finasteride.

[0156] Experimental methods

[0157] Cytotoxicity test

[0158] 1) Cell plating: Select primary human sebaceous gland cells that have been revived and passaged for more than 2 generations for the experiment. Discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off the culture flask under a microscope, add 1 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 5 mL of complete culture medium and resuspend to count the cells. Dilute the cell suspension to a density of 1.5×10 5Each well was inoculated into a 24-well plate at 100 μL / well and then placed in a 37°C, 5% CO2 incubator for overnight culture.

[0159] Sample addition: Take out the 96-well plate, discard the supernatant, add complete medium to the normal control group, add complete medium containing samples of different concentrations to the sample group, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 24 h;

[0160] 3) Cell viability detection: Take out the 96-well plate, place it for about 10 minutes to equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, and then detect the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula: Cell viability (%) = sample OD value / blank OD value × 100%

[0161] Subsequent efficacy evaluation experiments were conducted on samples with a safe concentration of more than 90% cell viability.

[0162] 3.2 Efficacy evaluation

[0163] 1) Cell plating: discard the culture medium in the T25 culture flask, wash twice with PBS, add 1 mL of trypsin containing 0.25% EDTA, and digest in a CO2 incubator for 2 minutes. After observing the cells fall off under a microscope, add 5 mL of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 minutes, and discard the supernatant. Add 5 mL of complete culture medium and resuspend to count the cells. Dilute the cell suspension to a density of 1.2×10 5 Each well was inoculated into a 24-well plate, 1 mL / well, and then placed in a 37°C, 5% CO2 incubator for overnight culture.

[0164] Note: A blank control group, a test sample group and a positive control group were set up for each test, and three replicate wells were set up for each group.

[0165] 2) Sample addition: After 24 hours of culture, the cell culture medium was replaced with the culture medium containing the test sample, 1 mL / well, and culture was continued for 24 hours. The positive control group was a complete culture medium containing 0.5 mg / ml finasteride, and the model group was added with 0.1 μM DHT.

[0166] 3) Detection: discard the supernatant, wash the cells twice with PBS, then add 4% paraformaldehyde to fix the cells for 5-15 min, change the solution, and add 500 μL-1000 μL Nile red for staining for 15 min. Use a multifunctional microplate reader to detect the fluorescence intensity (IOD) of each well at 543 nm excitation and 598 nm emission wavelength, and calculate the lipid droplet formation inhibition rate (%) based on the relative IOD.

[0167] 4) Calculation of results: The calculation formula is as follows: Lipid droplet formation inhibition rate % = (model group IOD-sample group IOD) / (model group IOD-solvent well IOD) × 100%

[0168] 5) Data statistics: Graphpad Prism software was used to plot experimental data, and one-way ANOVA was used to analyze data between multiple groups, or t-test was used to compare data between groups. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference.

[0169] Experimental Results

[0170] As can be seen from Table 5, compared with the blank control BC, DHT can significantly increase the Nile red fluorescence intensity (IOD) of sebaceous gland cell lipid droplets, with a significant difference (P<0.05); compared with the model group, 50μg / ml finasteride can significantly reduce IOD, with an inhibition rate of 39.13% (P<0.01), both indicating that the model is reliable; compared with the model group, 1000μg / ml hydrangea polysaccharide can significantly increase IOD, with an increase rate of 15.53% (P<0.05), indicating that hydrangea polysaccharide has the potential to promote sebum production or moisturize; 3% glycerol has no significant effect on sebum production.

[0171] Table 5. Effects of Hydrangea polysaccharide on lipid droplets in sebaceous gland cells

[0172]

[0173] Compared with the blank control BC, # P<0.01; compared with the model group, *P<0.05

[0174] As can be seen from Table 6, compared with the blank control BC, 10-1000 μg / ml Hydrangea polysaccharide can significantly increase IOD, with the increase rates being 28.66% (P<0.001), 22.24% (P<0.01) and 26.18% (P<0.01), respectively, indicating that Hydrangea polysaccharide has the effect of promoting sebum production, or nourishing and moisturizing; 3% glycerol has no significant effect on sebum production.

[0175] Table 6. Effects of Hydrangea polysaccharide on lipid droplets in sebaceous gland cells

[0176]

[0177] Compared with blank control BC, **P<0.01, ***P<0.001

[0178] Experimental conclusion

[0179] Under the conditions of this experiment, the polysaccharide extract of Hydrangea spp. significantly promoted sebum secretion in the concentration range of 10 μg / ml-1 mg / ml, had moisturizing or nourishing effects, and its effect was better than 3% glycerol.

[0180] Example 6: Anti-wrinkle effect of Hydrangea polysaccharide

[0181] Materials and methods

[0182] Cells: HFF-1 human skin fibroblasts.

[0183] Main reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), TGF-β (MCE), Collagen I ELISA kit (Huamei Biotechnology).

[0184] Main equipment: CO2 incubator (healforce), biological safety cabinet (healforce), multifunctional microplate reader (Tecan).

[0185] Experimental method: The sample cytotoxicity experiment and efficacy evaluation test were carried out according to the "Q / YGEMG-SOP-2003 Human Fibroblast Type I Collagen Content Determination Operating Procedure" of the Basic Research Department.

[0186] Cytotoxicity test of HFF-1 cells

[0187] 1) Inoculate cells in 96-well plates (1×10 4 / well) and incubated overnight in a 37°C, 5% CO2 incubator.

[0188] 2) Administration: Remove the culture medium, add 100 μL of culture medium to each well of the blank control group, and add 100 μL of culture medium containing the sample of corresponding concentration to each well of the sample group, and culture in a 37° C., 5% CO2 incubator for 24 h.

[0189] 3) Detection: Take out the 96-well plate, place it for about 10 minutes, equilibrate to room temperature, add CCK8 detection reagent, 10 μL / well, incubate at 37°C for 1 hour, read the OD value at 450nm (minus the OD value at 600nm); Cell viability calculation formula:

[0190] Cell viability (%) = OD value of sample group / OD value of blank control group × 100%

[0191] Note: Subsequent efficacy evaluation tests were performed when the cell viability was above 90%.

[0192] Determination of relative content of type Ⅰ collagen in HFF-1 human skin fibroblasts

[0193] 1) Inoculate cells in 96-well plates (1×10 4 / well) and incubated overnight in a 37°C, 5% CO2 incubator.

[0194] 2) Administration: Remove the culture medium, set up 3 replicates in each group, add 150 μL of culture medium to each well of the blank control group (BC), the negative control group (NC) contains physiological saline, the positive control group (PC) adds culture medium containing TGF-β100 ng / mL, and the sample group adds culture medium containing the sample to be tested at the corresponding concentration. Culture for 24 hours.

[0195] 3) Detection: After 24 hours of culture, the supernatant was centrifuged and then detected using an enzyme-linked immunosorbent assay kit to calculate the average protein content.

[0196] 4) Calculation of improvement rate: Improvement rate (%) = (sample group - blank control group) / blank control group × 100% Statistical analysis

[0197] The mean value (Mean) and standard deviation (SD) of each group of experiments were calculated, and Graphpad Prism software was used for data statistical analysis. P < 0.05 indicated a significant difference, P < 0.01 indicated an extremely significant difference, and P > 0.05 indicated no significant difference.

[0198] Experimental Results

[0199] The cytotoxicity CCK8 test results of each sample are shown in Table 7.

[0200] Table 7. Sample cytotoxicity CCK8 test results

[0201]

[0202] According to the CCK8 results, the sample β-glucan and hydrangea polysaccharide extracts had no cytotoxicity in the concentration range of 0.13% to 0.50%.

[0203] Anti-wrinkle efficacy test results: see Table 8.

[0204] As shown in Table 8, compared with the blank control group, the positive control significantly promoted the secretion of type I collagen (p<0.001), and had anti-wrinkle effect; compared with the blank control:

[0205] 1) When β-glucan was present at 0.50%, 0.25%, and 0.13% concentrations, the average contents of type Ⅰ collagen were 2076.50±240.35ng / mL (P<0.001), 981.50±31.86ng / mL (P<0.001), and 804.50±43.79ng / mL (P<0.01), respectively;

[0206] 2) At the concentrations of 0.50%, 0.25% and 0.13% of hydrangea polysaccharide extract, the average contents of type I collagen were 2134.50±43.33ng / mL (P<0.01), 1310.50±83.05ng / mL (P<0.01) and 1253.00±208.12ng / mL (P<0.05), respectively.

[0207] Table 8. Average content of type Ⅰ collagen in samples

[0208]

[0209] Note: Comparison between sample group and blank group, ***p<0.001; **p<0.01; *P<0.05.

[0210] Experimental conclusion:

[0211] Under the conditions of this experiment, β-glucan and hydrangea polysaccharide extracts at a concentration of 0.13%-0.5% significantly promoted the secretion of type I collagen and had anti-wrinkle effects.

[0212] Example 7: Safety test of hydrangea polysaccharide

[0213] Experimental materials: SPF-grade fertilized chicken embryos of Bailaihang chicken, sodium chloride (Solarbio), sodium hydroxide (Shanghai McLean Co., Ltd.)

[0214] Test method: Based on SN / T2329-2009 "Chicken embryo chorioallantoic membrane test for eye irritation / corrosion of cosmetics"

[0215] Through the endpoint evaluation method, 0.3 ml of the sample was dripped onto the CAM. After 3 minutes of action, the test substance on the CAM membrane was gently rinsed with physiological saline to observe the bleeding, coagulation, and vascular dissolution, and photographed. At the same time, a negative control of 0.9% NaCl and a positive control of 0.2%, 0.3%, and 0.5% sodium hydroxide solution were set. The test results are shown in Figures 1-2 As shown, the sample was tested by the endpoint evaluation method. After 3 minutes of the sample action: compared with the negative control group, no obvious bleeding, coagulation, and vascular fusion were observed in the CAM at the concentrations of 10 mg / ml and 5 mg / ml of lily peptide, so the ES score of the sample hydrangea polysaccharide at the concentrations of 10 mg / ml and 5 mg / ml was 0. Under this test condition, hydrangea polysaccharide was non-irritating at the concentrations of 10 mg / ml and 5 mg / ml.

[0216] Example 8: Preparation of lip care product (lipstick) containing hydrangea polysaccharide

[0217] The formula is shown in Table 9. (1) Dry-mix the magnesium aluminum silicate and hydrangea polysaccharide of component A, then slowly add them into purified water and stir them continuously until uniform; (2) Mix components A and B, and heat them to 65-70°C; (3) Grind component C and add it to component B, and heat it to 70°C; (4) Add component E to the water phase, then add the oil phase, emulsify them, continue stirring, and cool; (5) Press molding to prepare a lip care product.

[0218] Table 9

[0219]

[0220]

[0221] Example 9: Preparation of lip care product (lip gloss) containing hydrangea polysaccharide

[0222] The formula is shown in Table 10. (1) Heat the A phase material to 65°C, stir evenly and homogenize for 2-3 minutes; (2) Add the B component, stir evenly and cool; (3) Press mold to prepare a lip care product.

[0223] Table 10

[0224]

Claims

1. Use of Hydrangea polysaccharide in lip care products.

2. The use according to claim 1, characterized in that The lip care is achieved by promoting the proliferation of lip epithelial cells.

3. The use according to claim 2, characterized in that: The mass fraction of the hydrangea polysaccharide in the lip care product is 0.001% to 5%. Preferably, the mass fraction of the hydrangea polysaccharide in the lip care product is 0.1%.

4. The use according to claim 1, characterized in that The hydrangea polysaccharide is obtained by crushing hydrangea fruiting bodies, adding liquid to dissolve, hot extraction, filtering, and ethanol precipitation.

5. The use according to claim 4, characterized in that The material-liquid ratio of the added liquid for dissolution is 1:20 (g / mL) to 1:80 (g / mL).

6. A lip care product comprising hydrangea polysaccharide, oil and wax substances, wherein: The mass fraction of the oil and wax substances in the whole lip care product is greater than 30%.

7. The lip care product according to claim 6, characterized in that: The mass fraction of the oil and wax substances in the whole lip care product is greater than 50%.

8. The lip care product according to claim 6, characterized in that: The lip care product is lipstick, lip gloss, lip cream, lip glaze, lip balm, or lip mask.

9. The lip care product according to claim 8, characterized in that: The lip care product further comprises at least one of a flavor, an antioxidant, an emollient, a filler, a colorant, a preservative, a binder, or a thickener.

10. The lip care product according to claim 9, characterized in that: The lip care product is a lipstick, comprising isooctyl palmitate, phenyl isopropyl polydimethicone, plant lanolin, octyldodecanol, polyisobutylene, PEG-10 polydimethicone, olive oil, silicon dioxide, bentonite, deionized water, hydrangea polysaccharide, 1,3-butylene glycol, disodium EDTA, color paste, antioxidant, preservative, and flavor; or The lip care product is a lip gloss, including glyceryl diisostearate, macadamia oil, No. 70 white oil, propyl dimethicone, vitamin E, glyceryl isooctanoate, polydimethylsiloxane, refined ozokerite, lanolin hydroxystearate, microcrystalline wax, essence, red iron oxide, refined water, and hydrangea polysaccharide.

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