New use of a long-stalked ilex glycoside in skin care products and active compositions and skin care products

Abstract

The present application relates to a kind of long stem ilex ginseng new use in skin care product and active composition and skin care product, product is emulsion, cream, water agent, spray, gel and other skin care products, the components of emulsion include: active composition, humectant, emollient, emulsifier, thickening agent and deionized water, the mass percentage content of active composition in skin care product is 0.005%-0.1%, active composition includes long stem ilex ginseng and wujin ginseng, the mass ratio of long stem ilex ginseng and wujin ginseng is 5:1-1:1, long stem ilex ginseng has at least one of the following effects: (1) UVB light damage pre-protection to skin;(2) after UVB light damage repair to skin;(3) up-regulate the trichohyalin gene expression in skin cell.The long stem ilex ginseng of the present application has the effects of UVB light damage pre-protection to skin, after UVB light damage repair to skin and up-regulating the trichohyalin gene expression in skin cell to repair skin barrier.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetics, more particularly, it relates to a new use of pedunculoside in skin care products, as well as active compositions and skin care products. BACKGROUND

[0002] Pedunculoside is extracted from the bark of Ilex rotunda Thunb. The preparation method of pedunculoside has been reported in a large number of prior art documents. For example, Chinese patent CN109970838B (a preparation method of pedunculoside) reports that Jiebiyang is extracted by heating reflux with ethanol water, impurities are washed and eluted by polyamide column adsorption, and finally pedunculoside is obtained by recrystallization with 30%-60% ethanol; Chinese patent publication CN102643322A (a method for simultaneously preparing pedunculoside and syringin) reports that Jiebiyang is separated by macroporous resin adsorption after solvent extraction, and then high-purity pedunculoside and syringin are obtained by ODS column chromatography or Sephadex LH-20 column chromatography, and preparative liquid chromatography; Chinese patent publication CN103351419A reports a two-step method for simultaneously preparing pedunculoside and syringin, Jiebiyang is extracted by refluxing with 20%-70% ethanol water, the filtrate and the residue are collected, the residue is dried, and the monomer of pedunculoside is obtained after purification. In addition, the use of pedunculoside in anti-inflammatory, anticancer, and antiviral aspects has also been reported in existing documents, see patents CN105125567A, CN114558023A, and CN107095878B. However, in the existing reports, pedunculoside and its compositions have not been found to be used in skin sunscreen repair products for UVB photodamage protection, UVB photodamage repair, and skin barrier repair.

[0003] According to relevant data, ultraviolet (UV) radiation is a general term for electromagnetic radiation with wavelengths ranging from 10 to 400 nm. Based on their wavelengths, UV radiation can be divided into long-wave ultraviolet (315-400 nm, UVA), medium-wave ultraviolet (280-315 nm, UVB), and short-wave ultraviolet (200-280 nm, UVC). UVC has the highest energy but is blocked by the ozone layer and usually does not reach the Earth's surface. UVB, on the other hand, is the main cause of sunburn and skin redness. Although UVB is easily absorbed by the stratospheric ozone layer, with the depletion of the ozone layer, sunburn and skin redness caused by UVB are receiving increasing attention. Currently, although commercially available skin sun protection and repair products contain ingredients that can improve photochromic and degenerative changes and wrinkles, such as benzophenone, ethylhexyl salicylate, aromatic compounds, p-aminobenzoic acid derivatives, benzyl derivatives, cinnamic acid derivatives, and benzotriazole derivatives, most of these ingredients are derived from chemical synthesis, and no plant-derived holmyl glycosides have been found for similar applications.

[0004] Therefore, there is an urgent need to propose a new use for Ilex chinensis glycosides in skincare products, as well as active compositions and skincare products that utilize plant-derived Ilex chinensis glycosides to protect the skin from UVB photodamage, repair the skin after UVB photodamage, and repair the skin barrier. This would fill the current technological gap in the application of Ilex chinensis glycosides in skincare products for UVB photodamage protection, UVB photodamage repair, skin barrier repair, and in sun protection and repair products. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the main objective of this invention is to propose a new use of holly glycosides in skincare products, as well as an active composition and skincare product. This aims to solve the current technical problem of not utilizing holly glycosides in skin sun protection and repair products for pre-UVB photodamage protection, post-UVB photodamage repair, and skin barrier repair.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a novel use of holly glycosides in skincare products, wherein the holly glycosides possess at least one of the following effects:

[0008] (1) Protect skin keratinocytes from UVB photodamage;

[0009] (2) Repairing skin keratinocytes after UVB light damage;

[0010] (3) Upregulate the expression of the scutellarin gene in skin keratinocytes.

[0011] It should be noted that loricrin is a major component of the keratinized capsule of the final differentiated keratinocytes and plays an important role in the barrier function of the epidermis.

[0012] Preferably, the protection of skin keratinocytes against UVB photodamage by the ideologian oleuropein includes: in a photoprotection model, under the condition that the concentration of ideologian oleuropein sample is 15.625-250 μg / mL, the viability of skin keratinocytes increases by 34.22%-51.04% compared with that without the addition of ideologian oleuropein.

[0013] Preferably, the repair of skin keratinocytes after UVB photodamage by the ide leucocephalic glycoside includes: in an anti-photoaging model, under the condition that the concentration of ide leucocephalic glycoside sample is 15.625-250 μg / mL, the skin keratinocyte activity increases by 31.19%-50.82% compared with that without the addition of ide leucocephalic glycoside.

[0014] Preferably, the upregulation of the leucocele glycoside in the expression of the leucocele protein gene in skin keratinocytes includes: in a skin barrier model, under the condition that the concentration of the leucocele glycoside sample is 15.625-250 μg / mL, the upregulation rate of the leucocele protein gene is 122.8%-242.56%.

[0015] Secondly, the present invention provides an active composition,

[0016] The active composition comprises holly glycoside and quinoin, wherein the mass ratio of holly glycoside to quinoin is 5:1 to 1:1, and the active composition possesses at least one of the following effects:

[0017] (1) Protect skin keratinocytes from UVB photodamage;

[0018] (2) Repairing skin keratinocytes after UVB light damage;

[0019] (3) Upregulate the expression of the scutellarin gene in skin keratinocytes.

[0020] Preferably, the mass ratio of long-stemmed holly glycoside to quinoa glycoside is 3:1.

[0021] Thirdly, the present invention provides a skin care product, the type of which includes: lotion, cream, liquid, spray or gel, wherein the active composition in the skin care product has a mass percentage content of 0.005%-0.1%.

[0022] Preferably, the active composition in the skin care product has a mass percentage of 0.01-0.095%, more preferably 0.015-0.09%, more preferably 0.02-0.085%, more preferably 0.025-0.08%, more preferably 0.03-0.075%, more preferably 0.035-0.07%, more preferably 0.04-0.065%, more preferably 0.045-0.06%, more preferably 0.05-0.055%, and more preferably 0.05%.

[0023] Preferably, when the skin care product is a lotion, the ingredients of the lotion further include: moisturizer, emollient, emulsifier, thickener, and deionized water.

[0024] Preferably, the moisturizer in the emulsion is selected from any two or more combinations of dipropylene glycol, panthenol, and glyceryl polyether-26.

[0025] Preferably, the emulsion contains a moisturizer selected from any one or a combination of more than one of polydimethylsiloxane, triglyceride (ethylhexanoate), petrolatum, and hydrogenated polyisobutylene.

[0026] Preferably, the emulsifier in the emulsion is selected from any one or a combination of more than one of stearyl alcohol polyether-21, potassium cetyl phosphate, sucrose polystearate, and cetearyl olive oil ester.

[0027] Preferably, the thickener in the emulsion includes sodium alginate.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] (1) The present invention provides a new use of long-stemmed holly glycoside in skin care products. The long-stemmed holly glycoside has the effects of protecting skin keratinocytes before UVB light damage, repairing skin keratinocytes after UVB light damage, and upregulating the expression of the stigma protein gene in skin keratinocytes to repair the skin barrier.

[0030] (2) The present invention provides a skin care product comprising an active composition, wherein the active composition comprises holly glycoside and quinoin, wherein the mass ratio of holly glycoside to quinoin is 5:1-1:1. The skin care product containing the active composition exhibits excellent photoprotection, photodamage repair, and skin barrier repair effects. Compared to holly glycoside alone, a mass ratio of holly glycoside to quinoin at a sample concentration of 60 μg / mL, with the synergistic effect of holly glycoside and quinoin, demonstrates better effects in photoprotection, photodamage repair, and skin barrier repair. It can significantly protect keratinocytes from photodamage before light exposure to ensure continued cell proliferation, and can also significantly repair keratinocyte proliferation after light exposure, exhibiting excellent photoprotection and anti-photoaging effects. Furthermore, holly glycoside can upregulate the expression of the scleroderma protein gene to repair the skin barrier and protect damaged cells.

[0031] (3) The long-stemmed holly glycoside in the skin care product provided by the present invention can be extracted from plants, is suitable for industrial mass production, and can fill the technical gap of long-stemmed holly glycoside in protecting the skin before UVB light damage, repairing the skin after UVB light damage, and repairing the skin barrier. Attached Figure Description

[0032] Figure 1 The image shows the cell morphology under photoprotection in Test Example 1 of this application, which is either a monomer of Ilex chinensis or a combination of Ilex chinensis and ginsenoside.

[0033] Figure 2 The image shows the cell viability of the holly glycoside monomer or the holly glycoside and ginseng combination in Test Example 1 of this application under a photoprotection model using a CCK-8 assay.

[0034] Figure 3 This is a cell morphology diagram of the anti-photoaging model of the holly glycoside monomer or the combination of holly glycoside and quinoin in Test Example 2 of this application.

[0035] Figure 4 The image shows the cell viability of CCK-8 assay cells in the anti-photoaging model of Ilex chinensis glycoside and Ilex chinensis glycoside composition in Test Example 2 of this application.

[0036] Figure 5 This diagram shows the relative expression levels of the lobe protein (LOR) gene in keratinocytes under a skin barrier model of either the lobe protein monomer or the combination of lobe protein and ginseng in Test Example 3 of this application. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified illustrations of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0038] The source information of the relevant raw materials and materials involved in the following test examples is as follows:

[0039] HaCaT cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences; culture conditions: DMEM medium, 10% FBS fetal bovine serum by volume);

[0040] The CCK-8 test kit is sourced from Xinbosheng Biotechnology Co., Ltd.

[0041] The glycosides in long-stemmed holly are derived from Yunnan Yunke Special Plant Extraction Laboratory Co., Ltd.

[0042] The ginsenosides are sourced from Yunnan Yunke Special Plant Extraction Laboratory Co., Ltd.

[0043] Test Example 1: In vitro photoprotection assessment experiment of keratinocytes

[0044] Prolonged exposure to ultraviolet (UV) radiation can easily cause age spots, wrinkles, and skin laxity, primarily due to UVB rays. Pre-UV exposure to a sample followed by UV irradiation, and then incubating the resulting keratinocytes for a certain period to assess their morphology and viability, is an important method for evaluating the photoprotective performance (i.e., pre-UVB damage protection efficacy) of the added drug.

[0045] 1.1 Establishment of photoprotection model and sample processing

[0046] Blank control group: 100 μL of well-shaped cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 The cells were cultured at a concentration of 5% per mL until they reached about 80% of the culture plate. Then, the cells were cultured at 37°C for 24 hours in CO2 at a volume percentage of 5%.

[0047] Model control group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 Cells / mL, when the cells have grown to about 80% of the culture plate, are then irradiated with UVB (light intensity 20 mJ / cm²). 2 After irradiation for 15 minutes (lighting distance: 10cm), the cells were cultured for 24 hours in CO2 at 37°C with a volume percentage of 5%.

[0048] Experimental group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 Cells / mL were collected, and when the cells reached approximately 80% confluence with the culture plate, different concentrations of holly glycoside monomer or a combination of holly glycoside and ginsenoside were added before light irradiation for 2 hours; then UVB irradiation (light intensity: 20 mJ / cm²) was applied. 2 After irradiation for 15 minutes (lighting distance: 10cm), the cells were cultured for 24 hours in CO2 at 37°C with a volume percentage of 5%.

[0049] Verification method:

[0050] A) Add 100 μL of cell suspension to the center of each 96-well cell culture plate, with a cell density of 1 × 10⁻⁶ cells. 5 The cell / mL cell suspension was prepared with complete culture medium; the 96-well cell culture plate was placed in an incubator and the cells were cultured for 24 hours at 37°C with a volume percentage concentration of 5% CO2.

[0051] B) Set up a blank control group, a model control group, and an experimental group. In the experimental group, the sample types according to the serial numbers shown in Table 2 were incubated separately, and a corresponding sample concentration was set for each sample type. Each sample type was pretreated for 2 hours under a light intensity of 20 mJ / cm². 2 The light exposure distance was 10cm, and the irradiation time was 15min.

[0052] C) Replace the cell culture plates of the blank control group, model control group and experimental group with complete culture medium containing CCK-8, incubate for 2 hours, and measure the absorbance at a wavelength of 450 nm using an ELISA reader to obtain the optical density value OD1 of the blank control group and the optical density value OD2 of the experimental group.

[0053] D) Experimental data were processed using GraphPad, and statistical analysis was performed using One-way ANDNA (and nonparametric) Turkey: Compare all pairs of columns. Each experiment was repeated at least 3 times. Data are expressed as Mean ± SEM. P < 0.05 indicates statistical significance. The results of appropriate sample concentrations (μg / mL) and corresponding keratinocyte viability values ​​are shown in Table 1.

[0054] Table 1. Sample types, composition, concentration, and viability of keratinocytes in the photoprotection model.

[0055]

[0056]

[0057] See Figure 1 The images show the cell morphology of the blank control group, the model group, and the experimental group (a combination of 60 μg / mL 1:1 mass ratio of 1,000 μg / mL 1,000 μg / mL 1,000 μg / mL 1,000 μg / mL 1,000 μg / mL 1,000 μg / mL 1) under photoprotection model.

[0058] See Figure 2 The cell viability in the model control group without added lecithin monomer was only about 40% of that in the blank control group. This indicates that after irradiation with UVB at a wavelength of 280-315nm for 15 minutes, the viability of keratinocytes decreased significantly after incubation for 24 hours.

[0059] In the experimental groups, holly glycoside monomers (containing concentrations of holly glycosides of 15.625 μg / mL, 31.25 μg / mL, 60 μg / mL, 125 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL in each group) were first added to the complete culture medium, which was then irradiated with UVB. The results showed that the experimental groups with added holly glycoside monomers (containing concentrations of 15.625 μg / mL, 31.25 μg / mL, 60 μg / mL, 125 μg / mL, 200 μg / mL, and 250 μg / mL in each group) exhibited a photoprotective effect before light irradiation, and could significantly protect the viability of keratinocytes. Among them, the experimental group with a sample concentration of 60 μg / mL of Ilex chinensis glycoside monomer had a cell viability that was 51.04% higher than that of the model control group. Its photoprotective effect of adding the drug before light exposure was the most prominent when the sample type only involved Ilex chinensis glycoside monomer.

[0060] Meanwhile, the combination of holly glycoside and ginseng (mass ratio 3:1) at a sample concentration of 60 μg / mL, when applied before light exposure, also significantly protected keratinocytes from further proliferation. The cell viability increased by 111.98% compared to the model control group. This value is higher than that of samples involving only holly glycoside monomers and samples involving the combination of holly glycoside and ginseng (with a holly glycoside concentration of 60 μg / mL) at other mass ratios. This indicates that the combination of holly glycoside and ginseng (mass ratio 3:1) has a particularly prominent synergistic effect and provides significant pre-light exposure photoprotection.

[0061] Test Example 2: In vitro keratinocyte UV damage repair experiment

[0062] 2.1 Establishment of anti-photoaging model and sample processing

[0063] Blank control group: 100 μL of well-shaped cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 The cells were cultured at a concentration of 5% per mL until they reached about 80% of the culture plate. Then, the cells were cultured at 37°C for 24 hours in CO2 at a volume percentage of 5%.

[0064] Model control group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 Cells / mL, when the cells have grown to about 80% of the culture plate, are then irradiated with UVB (light intensity 20 mJ / cm²). 2 After irradiation for 15 minutes (lighting distance: 10cm), the cells were cultured for 24 hours at 37°C with CO2 at a volume percentage concentration of 5%.

[0065] Experimental group: 100 μL of well-morphologically healthy cells were added to a 96-well cell culture plate, with a cell suspension density of 1 × 10⁻⁶ cells / well. 5 Cells / mL, when the cells have grown to about 80% of the culture plate, are irradiated with UVB (light intensity: 20 mJ / cm²). 2 After irradiation for 15 minutes (lighting distance: 10cm), different concentrations of Ilex longiflorum glycoside monomer or a combination of Ilex longiflorum glycoside and ginsenoside were added, and the cells were cultured for 24 hours at 37°C with CO2 at a volume percentage concentration of 5%.

[0066] Verification method:

[0067] A) Add 100 μL of cell suspension to the center of each 96-well cell culture plate, with a cell density of 1 × 10⁻⁶ cells. 5 The cell count was 10 ...

[0068] B) Set up a blank control group, a model control group, and an experimental group, and irradiate with UVB light at an intensity of 20 mJ / cm². 2 The light distance was 10cm. After 15 minutes of light exposure, the test samples in the experimental group were incubated with different sample types in different number groups as shown in Table 3. Different sample concentrations were set for each sample type, and the cells were cultured for 24 hours at 37°C with CO2 at a volume percentage concentration of 5%.

[0069] C) Replace the cell culture plates of the blank control group, model control group and experimental group with complete medium containing CCK-8, incubate for 2 hours, and measure the absorbance at a wavelength of 450 nm using an ELISA reader to obtain the optical density value OD1 of the blank control group and the optical density value OD2 of the experimental group.

[0070] D) Experimental data were processed using GraphPad, and statistical analysis was performed using One-way ANDNA (and nonparametric) Turkey: Compare all pairs of columns. A blank control group, a model control group, and an experimental group were set up. Each experiment was repeated at least 3 times. Data are expressed as Mean±SEM. P<0.05 indicates statistical significance. The sample concentration (μg / mL) and the corresponding keratinocyte viability values ​​are shown in Table 2.

[0071] Table 2. Sample types, composition, concentration, and viability of keratinocytes in the anti-aging model.

[0072]

[0073]

[0074] See Figure 3 The figures show the cell morphology of the blank control group, the model group, and the experimental group (a combination of 60 μg / mL 1:1 of holly glycoside and oolong glycoside, 60 μg / mL holly glycoside monomer, and 300 μg / mL holly glycoside monomer) under the anti-photoaging model.

[0075] See Figure 4 Meanwhile, after 15 minutes of UVB irradiation, the cell viability in the culture dish of the model control group was 50% lower than that of the blank control group.

[0076] In the experimental group, after UVB irradiation, samples of pre-dissolved holly glycoside monomers (concentrations of 15.625 μg / mL, 31.25 μg / mL, 60 μg / mL, 125 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL) or a combination of holly glycosides and quinoin were added to the complete culture medium. After incubation for 24 hours, it was found that the concentration of holly glycoside monomers at sample concentrations of 15.625 μg / mL, ... Under conditions of 31.25 μg / mL, 60 μg / mL, 125 μg / mL, 200 μg / mL, and 250 μg / mL, the sample exhibited keratinocyte survival and repair effects. Among these, the experimental group with a sample concentration of 60 μg / mL of Ilex chinensis glycoside monomer showed 50.82% higher cell viability than the model control group. The effect of photo-induced keratinocyte repair (i.e., the anti-aging effect of the sample) was most prominent when the sample only involved Ilex chinensis glycoside monomer.

[0077] Meanwhile, the combination of holly glycoside and ginsenoside (mass ratio 3:1) under light irradiation at a sample concentration of 60 μg / mL also significantly repaired keratinocytes, with cell viability increasing by 100.39% compared to the model control group. This value is higher than that of samples involving only holly glycoside monomers and samples involving holly glycoside and ginsenoside combination at other mass ratios. This indicates that the combination of holly glycoside and ginsenoside (mass ratio 3:1) has a particularly prominent synergistic effect and plays a significant anti-aging role after light irradiation (keratinocytes continue to proliferate).

[0078] Test Example 3: In vitro evaluation experiment of skin barrier function using keratinocytes

[0079] 3.1 Skin barrier model establishment and sample processing

[0080] The blank control group, model control group, experimental group and test example 1 are identical.

[0081] Verification method:

[0082] A) Add 100 μL of cell suspension to the center of each 96-well cell culture plate, with a cell density of 1 × 10⁻⁶ cells. 5 The cell / mL cell suspension was prepared with complete culture medium and cultured at 37°C for 24 hours with 5% CO2 (v / v).

[0083] B) A blank control group, a model control group, and an experimental group were set up. In the experimental group, samples were incubated at a concentration permissible for cytotoxicity. The different sample types listed in Table 4 were used for incubation, with different concentrations set for each sample type. Pretreatment lasted 2 hours, followed by UVB irradiation at a light intensity of 20 mJ / cm². 2 And the light distance is 10cm;

[0084] C) RNA extraction, RNA content determination, reverse transcription of cDNA, and quantitative real-time PCR to test gene expression levels;

[0085] D) Result Calculation

[0086] The calculation method used was Comparative Cт(ΔΔCт), with a model control group and β-actin as the internal reference gene. RQ = 2. -ΔΔCt Where ΔΔCт=ΔCt treated -ΔCt control ,ΔCt treated and ΔCt control These are the Ct differences between the target gene and the reference gene in the experimental group and the model control group, respectively, i.e., ΔCt = CT. target -CT β-actinThe RQ value represents the relative expression level of the target gene mRNA.

[0087] The upregulation rate of lobe protein gene (LOR) expression was calculated using the following formula.

[0088] Upward adjustment rate % = (RQ treated -RQ control ) / RQ blank， *100%

[0089] E) Result Calculation

[0090] Experimental data were processed using GraphPad, and statistical analysis was performed using One-way ANDNA (and nonparametric) Turkey: Compare all pairs of columns. Each experiment was repeated at least three times. Data are expressed as Mean ± SEM, and P < 0.05 was considered statistically significant. The sample concentrations (μg / mL) and the corresponding upregulation rates of LOR expression are shown in Table 3.

[0091] Table 3. Sample types, composition, concentration, and upregulation rate of lobe protein gene (LOR) expression in keratinocytes in the skin barrier model.

[0092]

[0093]

[0094] The specific test results are shown below:

[0095] Depend on Figure 5It was found that before UVB irradiation, adding pre-dissolved holly glycoside monomers (sample concentrations of 15.625 μg / mL, 31.25 μg / mL, 60 μg / mL, 125 μg / mL, 200 μg / mL, and 250 μg / mL) or a combination of holly glycoside and ginseng (mass ratio of 3:1) and incubating, RNA was collected from cell samples and reverse transcribed to obtain cDNA. q-PCR was performed on the cDNA. The results showed that compared to the model control group (where the expression level of the lobe protein gene was 0.4 times that of the blank group), the upregulation rates of lobe protein gene (LOR) expression in the experimental groups at holly glycoside monomer concentrations of 15.625-250 μg / mL were 146.68%, 196.23%, 242.56%, 195.67%, 156.12%, and 122.28%, respectively. This fully demonstrates that the ide glycoside monomer has the effect of protecting the skin barrier, and the upregulation rate of the lobe protein gene (LOR) corresponding to the experimental group with a sample concentration of 60 μg / mL ide glycoside monomer was 242.56%, and its effect of protecting the skin barrier is most prominent when the sample type only involves ide glycoside monomer.

[0096] Meanwhile, the photoprotective effect of the combination of holly glycoside and ginseng (mass ratio of 3:1) in the pre-light-exposed sample at a dose of 60 μg / mL significantly repaired keratinocytes and upregulated the expression of the lobe protein gene. The upregulation rate of the corresponding lobe protein gene (LOR) expression was 297.43%, which was significantly higher than the 242.56% upregulation rate of the lobe protein gene (LOR) in the experimental group with a sample concentration of 60 μg / mL of holly glycoside monomer. This indicates that the combination of holly glycoside and ginseng (mass ratio of 3:1) has a synergistic effect and plays a more significant role in protecting the skin barrier.

[0097] Test Example 4: In vitro 3T3 neutral red uptake phototoxicity efficacy evaluation and verification

[0098] Test substance: a combination of long-stemmed holly glycoside and quinoin in a ratio of 3:1.

[0099] Verification method:

[0100] A) Add 100 μL of culture medium to the outer wells of a 96-well tissue culture plate (blank control), and add 100 μL of cell density (1×10⁶) to the remaining wells. 5 Cell suspension of cells / mL was cultured for 24 hours until the cells reached 80% of the culture plate. Two plates were prepared for each experiment, including the same series of test substance concentrations, solvent control, blank control and positive control (chlorpromazine hydrochloride). One plate was used to determine cytotoxicity (-Irr) and the other plate was used to determine phototoxicity (+Irr).

[0101] B) Remove the culture medium, gently rinse the cells once or twice with 150 μL EBSS or PBS, add 100 μL of buffer containing an appropriate concentration of the test substance or solvent to the well, and culture the cells for 1 hour.

[0102] C) Expose one of the plates to light (+Irr) at room temperature at 1.7 mW / cm². 2 The cells were irradiated with light through the lid of a 96-well plate for 50 minutes; at the same time, another plate was placed in a dark box without light (-Irr) for 50 minutes.

[0103] D) Remove the test solution and wash the cells twice thoroughly with 150 μL of LEBSS. Add 100 μL of culture medium and incubate overnight (18-22 h, 5%-7.5% CO2, 37°C).

[0104] E) Examine the cells under a phase contrast microscope and record the changes in cell morphology caused by the cytotoxicity of the test substance to eliminate experimental errors;

[0105] F) Neutral Red Absorption (NRU) Measurement: Cells absorb neutral red into lysosomes and vacuoles within living cells, which can be used as a quantitative indicator of cell number and viability. Wash cells once or twice with 150 μL of pre-warmed EBSS, add 100 μL of culture medium containing 50 μg / mL neutral red, and culture for 3 hours. Remove the neutral red culture medium, wash cells once or twice with 150 μL of EBSS, add 150 μL of neutral red desorption solution, and shake the 96-well plate thoroughly for 10 minutes until neutral red is extracted from the cells and a homogeneous solution is formed. Detect the absorption at 540 nm using a microplate reader. Use blank wells as a reference control.

[0106] Finally, the conclusions were drawn by discussing the different cases using the photoirritation factor (PIF) standard.

[0107] If the test substance is cytotoxic under light (+Irr) and not cytotoxic under no light (-Irr), and the cytotoxicity assay was performed to achieve the maximum test concentration (Cmax), the >PIF can be calculated using the following formula:

[0108] PIF = Cmax(-Irr) / IC50(+Irr)

[0109] The evaluation criteria are: if only one ">PIF" is obtained, then any PIF value > 1 can indicate "potential phototoxicity".

[0110] The test substance did not show cytotoxicity at the concentration (60 μg / mL; a combination of holly glycoside and quinoin = 3:1). The "PIF = *1" indicates that there is "no potential phototoxicity".

[0111] According to its experimental conditions, the calculated PIF value of idexin glycoside was *1, indicating no potential phototoxicity. This ensures that the dosage used in the product is safe.

[0112] The specific test results are shown below:

[0113] Test data shows that the concentration of holly glycoside in the sunscreen composition is 60 μg / mL, with a PIF of *1, indicating no potential phototoxicity. This ensures that the dosage used in the product is safe.

[0114] Test Example 5: Safety Verification Through Skin Irritation / Corrosion Testing

[0115] Test substance: Long-stemmed holly glycoside clear sunscreen product (the mass percentage of the active combination of long-stemmed holly glycoside and quinoin in the test substance is 0.005-0.1%. It should be noted that the determination of this content is based on the amplification of the corresponding data in the test case).

[0116] Verification method:

[0117] A) This invention is based on the white emulsion of the long stem;

[0118] B) The experimental animals used were New Zealand common grade rabbits, which were raised under conditions of 20℃-26℃ and relative humidity of 40%-70%.

[0119] C) Laboratory animals should be allowed at least 3 days to acclimatize to the laboratory environment. Approximately 24 hours before the experiment, the hair on both sides of the spine on the back of the laboratory animal should be shaved, taking care not to damage the epidermis. The shaved area should be 3cm × 3cm on each side. Apply approximately 0.5mL of the test substance directly to one side of the skin, covering an area of ​​2.5cm × 2.5cm. If the test substance is prepared using an irritating solvent, apply the solvent to the other side as a control. Apply once daily for 14 consecutive days. Starting from the second day, the hair should be shaved before each application, and any remaining test substance should be removed with water or a non-irritating solvent. Observe the results one hour later and score the skin reaction according to the "Cosmetic Safety Technical Specifications" (2015 edition). The control and test areas should be treated in the same way. Result evaluation: Calculate the average score per animal per day using the following formula to determine the intensity of skin irritation.

[0120]

[0121] The results of multiple skin irritation tests on rabbits are shown in Table 4:

[0122] Table 4. Results of repeated skin irritation tests on rabbits

[0123]

[0124]

[0125] Based on the results of multiple skin irritation tests on rabbits, it was found that the product containing long-stemmed wintergreen glycosides is non-irritating, proving that the product is safe and non-irritating to the skin.

[0126] Preparation Example 1

[0127] This example demonstrates the preparation of an emulsion with sun protection and repairing effects, the formulation of which is shown in Table 5:

[0128] Table 5. Emulsion formulations with sun protection and repair functions

[0129]

[0130]

[0131] Its preparation method is as follows:

[0132] (1) Add deionized water, humectant and thickener to the aqueous phase pot, homogenize and heat to 75°C, and keep warm for 10 minutes.

[0133] (2) Add the emulsifier to the emulsifier, homogenize and heat to 75°C, and keep warm for 10 minutes;

[0134] (3) Vacuum-draw the water pot and oil pot into the emulsifying pot, homogenize for 10 minutes, and then cool down.

[0135] (4) When the temperature drops to 40℃, add the sunscreen and repair active composition, stir evenly, and you will get the product.

[0136] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The use of holly glycoside in the preparation of skin care products, characterized in that, The long-stem holly glycoside possesses at least one of the following effects: (1) Protect skin keratinocytes from UVB photodamage; (2) Repairing skin keratinocytes after UVB photodamage; (3) Upregulate the expression of the stigma protein gene in skin keratinocytes for skin sun protection and repair.

2. The use according to claim 1, characterized in that, The protection of skin keratinocytes against UVB photodamage by the idemolecular osmanthus glycoside includes: in a photoprotection model, under the condition that the concentration of idemolecular osmanthus glycoside sample is 15.625-250 µg / mL, the viability of skin keratinocytes increases by 34.22%-51.04% compared with that without the addition of idemolecular osmanthus glycoside.

3. The use according to claim 1, characterized in that, The repair of skin keratinocytes after UVB photodamage by the stated holly glycoside includes: in an anti-photoaging model, under the condition that the concentration of holly glycoside sample is 15.625-250 µg / mL, the skin keratinocyte activity increases by 31.19%-50.82% compared with that without the addition of holly glycoside.

4. The use according to claim 1, characterized in that, The upregulation of the leucocytoside gene expression in skin keratinocytes by the leucocytoside includes: in a skin barrier model, under the condition that the concentration of leucocytoside sample is 15.625-250 µg / mL, the upregulation rate of the leucocytoside gene is 122.8%-242.56%.

5. An active composition, characterized in that, The active composition comprises holly glycoside and quinoin, wherein the mass ratio of holly glycoside to quinoin is 5:1 to 1:1, and the active composition possesses at least one of the following effects: (1) Protect skin keratinocytes from UVB photodamage; (2) Repairing skin keratinocytes after UVB photodamage; (3) Upregulate the expression of the stigma protein gene in skin keratinocytes.

6. The active composition according to claim 5, characterized in that, The mass ratio of long-stem holly glycoside to oolong glycoside is 3:

1.

7. A skincare product containing the active composition as described in claim 5, characterized in that, The types of skin care products include: lotions, creams, liquids, sprays, and gels, and the active composition in the skin care products has a mass percentage of 0.005%-0.1%.

8. The skincare product according to claim 7, characterized in that, When the skincare product is a lotion, the ingredients in the lotion also include: moisturizers, emollients, emulsifiers, thickeners, and deionized water.

9. The skincare product according to claim 8, characterized in that, The moisturizer in the emulsion is selected from any two or more combinations of dipropylene glycol, panthenol, and glyceryl polyether-26; the emollient in the emulsion is selected from any one or more combinations of polydimethylsiloxane, glyceryl tri(ethylhexanoate), petrolatum, and hydrogenated polyisobutylene.

10. The skincare product according to claim 8, characterized in that, The emulsifier in the emulsion is selected from any one or a combination of more than one of stearyl alcohol polyether-21, potassium cetyl phosphate, sucrose polystearate, and cetearyl olive oil ester; the thickener in the emulsion includes sodium alginate.

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